Modified pyrococcus polymerases and uses thereof

ABSTRACT

Provided herein are modified Archaeal family B polymerases derived from species of the Archaeal microorganism Pyrococcus that exhibit improved incorporation of nucleotide analogues utilized in DNA sequencing.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE

The Sequence Listing titled 051385-524F01US_SEQUENCE_LISTING_ST25.TXT,was created on Feb. 27, 2020 in machine format IBM-PC, MS-Windowsoperating system and is 95,424 bytes, is hereby incorporated byreference in its entirety for all purposes.

BACKGROUND

The present disclosure generally relates to modified polymerase enzymesthat exhibit improved incorporation of nucleotide analogues utilized inDNA sequencing.

DNA-polymerases add nucleotide triphosphate (dNTP) residues to the 3-endof the growing DNA chain, using a complementary DNA as template. Threemotifs, A, B and C, are seen to be conserved across all DNA-polymerases.Initial experiments with native DNA polymerases revealed difficultiesincorporating modified nucleotides. There are several examples in whichDNA polymerases have been modified to increase the rates ofincorporation of nucleotide analogues. For example, as described in WO2005/024010, modification to the amino acids at positions 408, 409, 410of a 9° N polymerase modifies the ability of polymerases to incorporatenucleotide analogues having a substituent at the 3′ position which islarger than a hydroxyl group (i.e., a reversible terminator).

Despite ongoing research, current modifications to the DNA polymerasestill do not show sufficiently high incorporation rates of modifiednucleotides. In nucleic acid sequencing applications, the modifiednucleotide typically has a reversible terminator at the 3′ position anda modified base (e.g., a base linked to a fluorophore via a cleavablelinker). In case of cleavable linkers attached to the base, there isusually a residual spacer arm left after the cleavage. This residualmodification may interfere with incorporation of subsequent nucleotidesby polymerase. Therefore, it is highly desirable to have polymerases forcarrying out sequencing by synthesis process (SBS) that are tolerable ofthese scars. In addition to rapid incorporation, the enzyme needs to bestable and have high incorporation fidelity. Balancing incorporationkinetics and fidelity can be a challenge. If the mutations in thepolymerase result in a rapid average incorporation half time but is toopromiscuous such that the inappropriate nucleotide is incorporated intothe primer, this will result in a large source of error in sequencingapplications. It is also desirable to design a polymerase capable toincorporating a variety of reversible terminators. Discovering apolymerase that has suitable kinetics and low misincorporation errorremains a challenge. Disclosed herein, inter alia, are solutions tothese and other problems in the art.

BRIEF SUMMARY

Provided herein are modified Archaeal family B polymerases derived fromthe Archaeal microorganism Pyrococcus.

In an aspect, provided herein is a polymerase including an amino acidsequence that is at least 80% identical to a continuous 500 amino acidsequence within SEQ ID NO: 1, including the following amino acids: analanine or serine at amino acid position 409 or an amino acid positionfunctionally equivalent to amino acid position 409; a glycine or alanineat amino acid position 410 or any amino acid that is functionallyequivalent to amino acid position 410; a proline, valine, glycine, orisoleucine at amino acid position 411 or any amino acid that isfunctionally equivalent to amino acid position 411; and an alanine atamino acid position 141 or an amino acid position functionallyequivalent to amino acid position 141; and an alanine at amino acidposition 143 or an amino acid position functionally equivalent to aminoacid position.

In an aspect, provided herein is a polymerase including an amino acidsequence that is at least 80% identical to a continuous 500 amino acidsequence within SEQ ID NO: 1, including the following amino acids: analanine or serine at amino acid position 409 or an amino acidfunctionally equivalent to amino acid position 409; a glycine at aminoacid position 410 or an amino acid functionally equivalent to amino acidposition 410; a proline, valine, glycine, isoleucine, or serine at aminoacid position 411 or an amino acid functionally equivalent to amino acidposition 411; an alanine at amino acid position 129 or an amino acidfunctionally equivalent to amino acid position 129; an alanine at aminoacid position 141 or an amino acid functionally equivalent to amino acidposition 141; an alanine at amino acid position 143 or an amino acidfunctionally equivalent to amino acid position 143; and an alanine atamino acid position 486 or an amino acid functionally equivalent toamino acid position 486.

In an aspect, provided herein is a polymerase including an amino acidsequence that is at least 80% identical to a continuous 500 amino acidsequence within SEQ ID NO: 1, including the following amino acids: analanine or serine at amino acid position 409 or an amino acid positionfunctionally equivalent to amino acid position 409; a glycine or alanineat amino acid position 410 or an amino acid position functionallyequivalent to amino acid position 410; a proline, valine, glycine, orisoleucine at amino acid position 411 or an amino acid positionfunctionally equivalent to amino acid position 411; an alanine at aminoacid position 141 or an amino acid position functionally equivalent toamino acid position 141; an alanine at amino acid position 143 or anamino acid position functionally equivalent to amino acid position 143;and a glutamic acid at amino acid position 153 or an amino acid positionfunctionally equivalent to amino acid position 153.

In an aspect, provided herein is a polymerase an amino acid sequencethat is at least 80% identical to a continuous 500 amino acid sequencewithin SEQ ID NO: 1, including the following amino acids: an alanine orserine at amino acid position 409 or an amino acid position functionallyequivalent to amino acid position 409; a glycine at amino acid position410 or an amino acid position functionally equivalent to amino acidposition 410; a proline, valine, glycine, isoleucine, or serine at aminoacid position 411 or an amino acid position functionally equivalent toamino acid position 411; and an alanine at amino acid position 129 or anamino acid functionally equivalent to amino acid position 129; analanine at amino acid position 141 or an amino acid functionallyequivalent to amino acid position 141; an alanine at amino acid position143 or an amino acid functionally equivalent to amino acid position 143;a glutamic acid at amino acid position 153 or an amino acid positionfunctionally equivalent to amino acid position 153; and an alanine atamino acid position 486 or an amino acid functionally equivalent toamino acid position 486.

Provided herein are methods of using modified Archaeal family Bpolymerases derived from the Archaeal microorganism Pyrococcus forimproved incorporation of modified nucleotides into a nucleic acidsequence.

In an aspect, provided herein is a method of incorporating a modifiednucleotide into a nucleic acid. The method includes allowing thefollowing components to interact: (i) a DNA template, (ii) a nucleotidesolution, and (iii) a polymerase, wherein the polymerase is a polymeraseof according to any one of the various embodiments described herein.

In an aspect, provided herein is a method of method of sequencing anucleic acid sequence including: a) providing a nucleic acid templatewith a primer hybridized to said template to form a primer-templatehybridization complex; b) adding a DNA polymerase and a nucleotidesolution to the primer-template hybridization complex, wherein the DNApolymerase is a polymerase of any one of the embodiments describedherein, and the nucleotide solution includes a modified nucleotide,wherein the modified nucleotide comprises a detectable label; c)subjecting primer-template hybridization complex to conditions whichenable the polymerase to incorporate a modified nucleotide into theprimer-template hybridization complex to form a modified primer-templatehybridization complex; and d) detecting the detectable label; therebysequencing a nucleic acid sequence.

In an aspect, provided herein is a kit including a polymerase asdescribed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C present data reporting average half time of incorporation ofmodified nucleotides bearing reversible terminator probes iso-1 andiso-2. Reactions were initiated in a buffer by the addition of 100 nMnucleotides (or 300 nM nucleotides for Challenge template sequences,unless otherwise indicated) and 133 nM DNA polymerase at a temperatureof 65° C. The data corresponds to the data presented in Table 4.

FIG. 2 presents data reporting average half time of incorporation ofmodified nucleotides bearing reversible terminator probes iso-1 andiso-2. Reactions were initiated in a buffer by the addition of 100 nMnucleotides (or 300 nM nucleotides for Challenge template sequences,unless otherwise indicated) and 133 nM DNA polymerase at a temperatureof 65° C. The data corresponds to the data presented in Table 4.

FIG. 3 shows the phylogenetic tree for related species. In the lowerleft corner, the 0.08 is used as a scale to estimate the geneticdistances between protein sequences. The genetic distance is an estimateof the divergence between two sequences (an estimate of the number ofmutations that have occurred since the two sequences shared a commonancestror). For example, the alignment between P. horikoshii and P.yayanosii shows 87% homology. The distance on the tree will give about0.13 divergence measuring only the horizontal branches between those twospecies.

DETAILED DESCRIPTION

Provided herein, are, for example, family B polymerases derived fromArchaea modified for improved incorporation of modified nucleotides intoa nucleic acid sequence and methods of use of the same.

I. Definitions

While various embodiments and aspects of the present invention are shownand described herein, it will be obvious to those skilled in the artthat such embodiments and aspects are provided by way of example only.Numerous variations, changes, and substitutions will now occur to thoseskilled in the art without departing from the invention. It should beunderstood that various alternatives to the embodiments of the inventiondescribed herein might be employed in practicing the invention.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in the applicationincluding, without limitation, patents, patent applications, articles,books, manuals, and treatises are hereby expressly incorporated byreference in their entirety for any purpose.

The abbreviations used herein have their conventional meaning within thechemical and biological arts. The chemical structures and formulae setforth herein are constructed according to the standard rules of chemicalvalency known in the chemical arts.

As used herein, the singular forms “a”, “an”, and “the” include pluralreferences unless the context clearly dictates otherwise.

As used herein, the term “about” means a range of values including thespecified value, which a person of ordinary skill in the art wouldconsider reasonably similar to the specified value. In embodiments, theterm “about” means within a standard deviation using measurementsgenerally acceptable in the art. In embodiments, about means a rangeextending to +/−10% of the specified value. In embodiments, about meansthe specified value.

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by a person of ordinaryskill in the art. See, e.g., Singleton et al., DICTIONARY OFMICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J. Wiley & Sons (New York,N.Y. 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL,Cold Springs Harbor Press (Cold Springs Harbor, N Y 1989). Any methods,devices and materials similar or equivalent to those described hereincan be used in the practice of this invention. The following definitionsare provided to facilitate understanding of certain terms usedfrequently herein and are not meant to limit the scope of the presentdisclosure.

“Nucleic acid” refers to nucleotides (e.g., deoxyribonucleotides orribonucleotides) and polymers thereof in either single-, double- ormultiple-stranded form, or complements thereof. The terms“polynucleotide,” “oligonucleotide,” “oligo” or the like refer, in theusual and customary sense, to a sequence of nucleotides. The term“nucleotide” refers, in the usual and customary sense, to a single unitof a polynucleotide, i.e., a monomer. Nucleotides can beribonucleotides, deoxyribonucleotides, or modified versions thereof.Examples of polynucleotides contemplated herein include single anddouble stranded DNA, single and double stranded RNA, and hybridmolecules having mixtures of single and double stranded DNA and RNA withlinear or circular framework. Non-limiting examples of polynucleotidesinclude a gene, a gene fragment, an exon, an intron, intergenic DNA(including, without limitation, heterochromatic DNA), messenger RNA(mRNA), transfer RNA, ribosomal RNA, a ribozyme, cDNA, a recombinantpolynucleotide, a branched polynucleotide, a plasmid, a vector, isolatedDNA of a sequence, isolated RNA of a sequence, a nucleic acid probe, anda primer. Polynucleotides useful in the methods of the disclosure maycomprise natural nucleic acid sequences and variants thereof, artificialnucleic acid sequences, or a combination of such sequences.

The term “duplex” in the context of polynucleotides refers, in the usualand customary sense, to double strandedness. Nucleic acids can be linearor branched. For example, nucleic acids can be a linear chain ofnucleotides or the nucleic acids can be branched, e.g., such that thenucleic acids comprise one or more arms or branches of nucleotides.Optionally, the branched nucleic acids are repetitively branched to formhigher ordered structures such as dendrimers and the like. Differentpolynucleotides may have different three-dimensional structures, and mayperform various functions, known or unknown.

Nucleic acids, including e.g., nucleic acids with a phosphothioatebackbone, can include one or more reactive moieties. As used herein, theterm reactive moiety includes any group capable of reacting with anothermolecule, e.g., a nucleic acid or polypeptide through covalent,non-covalent or other interactions. By way of example, the nucleic acidcan include an amino acid reactive moiety that reacts with an amino acidon a protein or polypeptide through a covalent, non-covalent or otherinteraction.

The terms “monophosphate” is used in accordance with its ordinarymeaning in the arts and refers to a moiety having the formula

The term “polyphosphate” refers to at least two phosphate groups, havingthe formula:

wherein np is an integer of 1 or greater and includes “diphosphate” and“triphosphate” with np=1 or 2 respectively. In embodiments, np is aninteger from 0 to 5. In embodiments, np is an integer from 0 to 2. Inembodiments, np is 2.

The term “base” as used herein refers to a purine or pyrimidine compoundor a derivative thereof, that may be a constituent of nucleic acid (i.e.DNA or RNA, or a derivative thereof). In embodiments, the base is aderivative of a naturally occurring DNA or RNA base (e.g., a baseanalogue). In embodiments, the base is a base-pairing base. Inembodiments, the base pairs to a complementary base. In embodiments, thebase is capable of forming at least one hydrogen bond with acomplementary base (e.g., adenine hydrogen bonds with thymine, adeninehydrogen bonds with uracil, guanine pairs with cytosine). Non-limitingexamples of a base includes cytosine or a derivative thereof (e.g.,cytosine analogue), guanine or a derivative thereof (e.g., guanineanalogue), adenine or a derivative thereof (e.g., adenine analogue),thymine or a derivative thereof (e.g., thymine analogue), uracil or aderivative thereof (e.g., uracil analogue), hypoxanthine or a derivativethereof (e.g., hypoxanthine analogue), xanthine or a derivative thereof(e.g., xanthine analogue), guanosine or a derivative thereof (e.g.,7-methylguanosine analogue), deaza-adenine or a derivative thereof(e.g., deaza-adenine analogue), deaza-guanine or a derivative thereof(e.g., deaza-guanine), deaza-hypoxanthine or a derivative thereof,5,6-dihydrouracil or a derivative thereof (e.g., 5,6-dihydrouracilanalogue), 5-methylcytosine or a derivative thereof (e.g.,5-methylcytosine analogue), or 5-hydroxymethylcytosine or a derivativethereof (e.g., 5-hydroxymethylcytosine analogue) moieties. Inembodiments, the base is thymine, cytosine, uracil, adenine, guanine,hypoxanthine, xanthine, theobromine, caffeine, uric acid, or isoguanine.In embodiments, the base is

A polynucleotide is typically composed of a specific sequence of fournucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine(T) (uracil (U) for thymine (T) when the polynucleotide is RNA). Thus,the term “polynucleotide sequence” is the alphabetical representation ofa polynucleotide molecule; alternatively, the term may be applied to thepolynucleotide molecule itself. This alphabetical representation can beinput into databases in a computer having a central processing unit andused for bioinformatics applications such as functional genomics andhomology searching. Polynucleotides may optionally include one or morenon-standard nucleotide(s), nucleotide analog(s) and/or modifiednucleotides.

The term “isolated”, when applied to a nucleic acid or protein, denotesthat the nucleic acid or protein is essentially free of other cellularcomponents with which it is associated in the natural state. It can be,for example, in a homogeneous state and may be in either a dry or anaqueous solution. Purity and homogeneity are typically determined usinganalytical chemistry techniques such as polyacrylamide gelelectrophoresis or high performance liquid chromatography. A proteinthat is the predominant species present in a preparation issubstantially purified.

The terms “analog” and “analogue”, in reference to a chemical compound,refers to compound having a structure similar to that of another one,but differing from it in respect of one or more different atoms,functional groups, or substructures that are replaced with one or moreother atoms, functional groups, or substructures. In the context of anucleotide useful in practicing the invention, a nucleotide analogrefers to a compound that, like the nucleotide of which it is an analog,can be incorporated into a nucleic acid molecule (e.g., an extensionproduct) by a suitable polymerase, for example, a DNA polymerase in thecontext of a dNTP analogue. The terms also encompass nucleic acidscontaining known nucleotide analogs or modified backbone residues orlinkages, which are synthetic, naturally occurring, and non-naturallyoccurring, which have similar binding properties as the referencenucleic acid, and which are metabolized in a manner similar to thereference nucleotides. Examples of such analogs include, include,without limitation, phosphodiester derivatives including, e.g.,phosphoramidate, phosphorodiamidate, phosphorothioate (also known asphosphothioate having double bonded sulfur replacing oxygen in thephosphate), phosphorodithioate, phosphonocarboxylic acids,phosphonocarboxylates, phosphonoacetic acid, phosphonoformic acid,methyl phosphonate, boron phosphonate, or O-methylphosphoroamiditelinkages (see Eckstein, OLIGONUCLEOTIDES AND ANALOGUES: A PRACTICALAPPROACH, Oxford University Press) as well as modifications to thenucleotide bases such as in 5-methyl cytidine or pseudouridine; andpeptide nucleic acid backbones and linkages. Other analog nucleic acidsinclude those with positive backbones; non-ionic backbones, modifiedsugars, and non-ribose backbones (e.g. phosphorodiamidate morpholinooligos or locked nucleic acids (LNA) as known in the art), includingthose described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters6 and 7, ASC Symposium Series 580, CARBOHYDRATE MODIFICATIONS INANTISENSE RESEARCH, Sanghui & Cook, eds. Nucleic acids containing one ormore carbocyclic sugars are also included within one definition ofnucleic acids. Modifications of the ribose-phosphate backbone may bedone for a variety of reasons, e.g., to increase the stability andhalf-life of such molecules in physiological environments or as probeson a biochip. Mixtures of naturally occurring nucleic acids and analogscan be made; alternatively, mixtures of different nucleic acid analogs,and mixtures of naturally occurring nucleic acids and analogs may bemade. In embodiments, the internucleotide linkages in DNA arephosphodiester, phosphodiester derivatives, or a combination of both.

The term “complement,” as used herein, refers to a nucleotide (e.g., RNAor DNA) or a sequence of nucleotides capable of base pairing with acomplementary nucleotide or sequence of nucleotides. As described hereinand commonly known in the art the complementary (matching) nucleoside ofadenosine is thymidine and the complementary (matching) nucleoside ofguanosine is cytidine. Thus, a complement may include a sequence ofnucleotides that base pair with corresponding complementary nucleotidesof a second nucleic acid sequence. The nucleotides of a complement maymatch, partially or completely, the nucleotides of the second nucleicacid sequence. Where the nucleotides of the complement completely matcheach nucleotide of the second nucleic acid sequence, the complementforms base pairs with each nucleotide of the second nucleic acidsequence. Where the nucleotides of the complement partially match thenucleotides of the second nucleic acid sequence, only some of thenucleotides of the complement form base pairs with nucleotides of thesecond nucleic acid sequence. Examples of complementary sequencesinclude coding and non-coding sequences, wherein the non-coding sequencecontains complementary nucleotides to the coding sequence and thus formsthe complement of the coding sequence. A further example ofcomplementary sequences are sense and antisense sequences, wherein thesense sequence contains complementary nucleotides to the antisensesequence and thus forms the complement of the antisense sequence.

As described herein the complementarity of sequences may be partial, inwhich only some of the nucleic acids match according to base pairing, orcomplete, where all the nucleic acids match according to base pairing.Thus, two sequences that are complementary to each other may have aspecified percentage of nucleotides that are complementary (i.e., about60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or higher complementarity over a specifiedregion).

“DNA” refers to deoxyribonucleic acid, a polymer of deoxyribonucleotides(e.g., dATP, dCTP, dGTP, dTTP, dUTP, etc.) linked by phosphodiesterbonds. DNA can be single-stranded (ssDNA) or double-stranded (dsDNA),and can include both single and double-stranded (or “duplex”) regions.“RNA” refers to ribonucleic acid, a polymer of ribonucleotides linked byphosphodiester bonds. RNA can be single-stranded (ssRNA) ordouble-stranded (dsRNA), and can include both single and double-stranded(or “duplex”) regions. Single-stranded DNA (or regions thereof) andssRNA can, if sufficiently complementary, hybridize to formdouble-stranded DNA/RNA complexes (or regions).

The term “DNA primer” refers to any DNA molecule that may hybridize to aDNA template and be bound by a DNA polymerase and extended in atemplate-directed process for nucleic acid synthesis.

The term “DNA template” refers to any DNA molecule that may be bound bya DNA polymerase and utilized as a template for nucleic acid synthesis.

The term “dATP analogue” refers to an analogue of deoxyadenosinetriphosphate (dATP) that is a substrate for a DNA polymerase. The term“dCTP analogue” refers to an analogue of deoxycytidine triphosphate(dCTP) that is a substrate for a DNA polymerase. The term “dGTPanalogue” refers to an analogue of deoxyguanosine triphosphate (dGTP)that is a substrate for a DNA polymerase. The term “dNTP analogue”refers to an analogue of deoxynucleoside triphosphate (dNTP) that is asubstrate for a DNA polymerase. The term “dTTP analogue” refers to ananalogue of deoxythymidine triphosphate (dUTP) that is a substrate for aDNA polymerase. The term “dUTP analogue” refers to an analogue ofdeoxyuridine triphosphate (dUTP) that is a substrate for a DNApolymerase.

The term “extendible” means, in the context of a nucleotide, primer, orextension product, that the 3′-OH group of the particular molecule isavailable and accessible to a DNA polymerase for extension or additionof nucleotides derived from dNTPs or dNTP analogues. “Incorporation”means joining of the modified nucleotide to the free 3′ hydroxyl groupof a second nucleotide via formation of a phosphodiester linkage withthe 5′ phosphate group of the modified nucleotide. The second nucleotideto which the modified nucleotide is joined will typically occur at the3′ end of a polynucleotide chain.

The term “modified nucleotide” refers to nucleotide or nucleotideanalogue modified in some manner. Typically, a nucleotide contains asingle 5-carbon sugar moiety, a single nitrogenous base moiety and 1 tothree phosphate moieties. In particular, embodiments, a nucleotide caninclude a blocking moiety or a label moiety. A blocking moiety on anucleotide prevents formation of a covalent bond between the 3′ hydroxylmoiety of the nucleotide and the 5′ phosphate of another nucleotide. Ablocking moiety on a nucleotide can be reversible (i.e., a reversibleterminator), whereby the blocking moiety can be removed or modified toallow the 3′ hydroxyl to form a covalent bond with the 5′ phosphate ofanother nucleotide. A blocking moiety can be effectively irreversibleunder particular conditions used in a method set forth herein. A labelmoiety of a nucleotide can be any moiety that allows the nucleotide tobe detected, for example, using a spectroscopic method. Exemplary labelmoieties are fluorescent labels, mass labels, chemiluminescent labels,electrochemical labels, detectable labels and the like. One or more ofthe above moieties can be absent from a nucleotide used in the methodsand compositions set forth herein. For example, a nucleotide can lack alabel moiety or a blocking moiety or both.

A “removable” group, e.g., a label or a blocking group or protectinggroup, refers to a chemical group that can be removed from a dNTPanalogue such that a DNA polymerase can extend the nucleic acid (e.g., aprimer or extension product) by the incorporation of at least oneadditional nucleotide. Removal may be by any suitable method, includingenzymatic, chemical, or photolytic cleavage. Removal of a removablegroup, e.g., a blocking group, does not require that the entireremovable group be removed, only that a sufficient portion of it beremoved such that a DNA polymerase can extend a nucleic acid byincorporation of at least one additional nucleotide using a dNTP of dNTPanalogue.

“Reversible blocking groups” or “reversible terminators” include ablocking moiety located, for example, at the 3′ position of thenucleotidide and may be a chemically cleavable moiety such as an allylgroup, an azidomethyl group or a methoxymethyl group, or may be anenzymatically cleavable group such as a phosphate ester. Suitablenucleotide blocking moieties are described in applications WO2004/018497, U.S. Pat. Nos. 7,057,026, 7,541,444, WO 96/07669, U.S. Pat.Nos. 5,763,594, 5,808,045, 5,872,244 and 6,232,465 the contents of whichare incorporated herein by reference in their entirety. The nucleotidesmay be labelled or unlabelled. They may be modified with reversibleterminators useful in methods provided herein and may be 3′-O-blockedreversible or 3′-unblocked reversible terminators. In nucleotides with3′-O-blocked reversible terminators, the blocking group —OR [reversibleterminating (capping) group] is linked to the oxygen atom of the 3′-OHof the pentose, while the label is linked to the base, which acts as areporter and can be cleaved. The 3′-O-blocked reversible terminators areknown in the art, and may be, for instance, a 3′-ONH₂ reversibleterminator, a 3′-O-allyl reversible terminator, or a 3′-O-azidomethyreversible terminator.

In embodiments, provided herein are polymerases capable of incorporatingthree differently sized reversible terminator probes linked to the 3′oxygen: an A-Term, S-Term, and i-term. A-Term refers to azide-containingterminators (Guo J, et al. PNAS 2008); for example having the formula:

S-Term refers to sulfide-containing terminators (WO 2017/058953); forexample having the formula

Wherein R″ is unsubstituted C₁-C₄ alkyl. The i-Term probe refers to anisomeric reversible terminator For example, an i-term probe has theformula:

wherein R^(A) and R^(B) are hydrogen or alkyl, wherein at least one ofR^(A) or R^(B) are hydrogen to yield a steroisomeric probe, and R^(C) isthe remainder of the reversible terminator. In embodiments, thenucleotide is

wherein Base is a Base as described herein, R³ is —OH, monophosphate, orpolyphosphate or a nucleic acid, and R′ is a reversible terminatorhaving the formula:

wherein R^(A) and R^(B) are hydrogen or alkyl and R^(C) is the remainderof the reversible terminator. In embodiments, the reversible terminatoris

In embodiments, the reversible terminator is

In embodiments, the reversible terminator is

In embodiments, the reversible terminator is

In embodiments, the reversible terminator is

In embodiments, the reversible terminator is

In embodiments, the reversible terminator is

In embodiments, the reversible terminator is

In embodiments, the reversible terminator is

In nucleotides with 3-unblocked reversible terminators, the terminationgroup is linked to the base of the nucleotide as well as the label andfunctions not only as a reporter by as part of the reversibleterminating group for termination of primer extension during sequencing.The 3′-unblocked reversible terminators are known in the art and includefor example, the “virtual terminator” as described in U.S. Pat. No.8,114,973 and the “Lightening terminator” as described in U.S. Pat. No.10,041,115, the contents of which are incorporated herein by referencein their entirety.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight (i.e., unbranched) or branchedcarbon chain (or carbon), or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include mono-, di- andmultivalent radicals. The alkyl may include a designated number ofcarbons (e.g., C₁-C₁₀ means one to ten carbons). Alkyl is an uncyclizedchain. Examples of saturated hydrocarbon radicals include, but are notlimited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl,t-butyl, isobutyl, sec-butyl, homologs and isomers thereof, for example,n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkylgroup is one having one or more double bonds or triple bonds. Examplesof unsaturated alkyl groups include, but are not limited to, vinyl,2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl,3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and thehigher homologs and isomers. An alkoxy is an alkyl attached to theremainder of the molecule via an oxygen linker (—O—). An alkyl moietymay be an alkenyl moiety. An alkyl moiety may be an alkynyl moiety. Analkyl moiety may be fully saturated. An alkenyl may include more thanone double bond and/or one or more triple bonds in addition to the oneor more double bonds. An alkynyl may include more than one triple bondand/or one or more double bonds in addition to the one or more triplebonds.

The term “alkylene,” by itself or as part of another substituent, means,unless otherwise stated, a divalent radical derived from an alkyl, asexemplified, but not limited by, —CH₂CH₂CH₂CH₂—. Typically, an alkyl (oralkylene) group will have from 1 to 24 carbon atoms, with those groupshaving 10 or fewer carbon atoms being preferred herein. A “lower alkyl”or “lower alkylene” is a shorter chain alkyl or alkylene group,generally having eight or fewer carbon atoms. The term “alkenylene,” byitself or as part of another substituent, means, unless otherwisestated, a divalent radical derived from an alkene.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcombinations thereof, including at least one carbon atom and at leastone heteroatom (e.g., O, N, P, Si, and S), and wherein the nitrogen andsulfur atoms may optionally be oxidized, and the nitrogen heteroatom mayoptionally be quaternized. The heteroatom(s) (e.g., N, S, Si, or P) maybe placed at any interior position of the heteroalkyl group or at theposition at which the alkyl group is attached to the remainder of themolecule. Heteroalkyl is an uncyclized chain. Examples include, but arenot limited to: —CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃,—CH₂—S—CH₂—CH₃, —CH₂—S—CH₂, —S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CHO—CH₃,—Si(CH₃)₃, —CH₂—CH═N—OCH₃, —CH═CH—N(CH₃)—CH₃, —O—CH₃, —O—CH₂—CH₃, and—CN. Two or three heteroatoms may be consecutive, such as, for example,—CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃. A heteroalkyl moiety may include oneheteroatom (e.g., O, N, S, Si, or P). A heteroalkyl moiety may includetwo optionally different heteroatoms (e.g., O, N, S, Si, or P). Aheteroalkyl moiety may include three optionally different heteroatoms(e.g., O, N, S, Si, or P). A heteroalkyl moiety may include fouroptionally different heteroatoms (e.g., O, N, S, Si, or P). Aheteroalkyl moiety may include five optionally different heteroatoms(e.g., O, N, S, Si, or P). A heteroalkyl moiety may include up to 8optionally different heteroatoms (e.g., O, N, S, Si, or P). The term“heteroalkenyl,” by itself or in combination with another term, means,unless otherwise stated, a heteroalkyl including at least one doublebond. A heteroalkenyl may optionally include more than one double bondand/or one or more triple bonds in additional to the one or more doublebonds. The term “heteroalkynyl,” by itself or in combination withanother term, means, unless otherwise stated, a heteroalkyl including atleast one triple bond. A heteroalkynyl may optionally include more thanone triple bond and/or one or more double bonds in additional to the oneor more triple bonds.

Similarly, the term “heteroalkylene,” by itself or as part of anothersubstituent, means, unless otherwise stated, a divalent radical derivedfrom heteroalkyl, as exemplified, but not limited by,—CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylenegroups, heteroatoms can also occupy either or both of the chain termini(e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, andthe like). Still further, for alkylene and heteroalkylene linkinggroups, no orientation of the linking group is implied by the directionin which the formula of the linking group is written. For example, theformula —C(O)₂R′— represents both —C(O)₂R′— and —R′C(O)₂—. As describedabove, heteroalkyl groups, as used herein, include those groups that areattached to the remainder of the molecule through a heteroatom, such as—C(O)R′, —C(O)NR′, —NR′R″, —OR′, —SR′, and/or —SO₂R′. Where“heteroalkyl” is recited, followed by recitations of specificheteroalkyl groups, such as —NR′R″ or the like, it will be understoodthat the terms heteroalkyl and —NR′R″ are not redundant or mutuallyexclusive. Rather, the specific heteroalkyl groups are recited to addclarity. Thus, the term “heteroalkyl” should not be interpreted hereinas excluding specific heteroalkyl groups, such as —NR′R″ or the like.

The terms “cycloalkyl” and “heterocycloalkyl,” by themselves or incombination with other terms, mean, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl,” respectively. Cycloalkyl andheterocycloalkyl are not aromatic. Additionally, for heterocycloalkyl, aheteroatom can occupy the position at which the heterocycle is attachedto the remainder of the molecule. Examples of cycloalkyl include, butare not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like. A “cycloalkylene” and a“heterocycloalkylene,” alone or as part of another substituent, means adivalent radical derived from a cycloalkyl and heterocycloalkyl,respectively.

In embodiments, the term “cycloalkyl” means a monocyclic, bicyclic, or amulticyclic cycloalkyl ring system. In embodiments, monocyclic ringsystems are cyclic hydrocarbon groups containing from 3 to 8 carbonatoms, where such groups can be saturated or unsaturated, but notaromatic. In embodiments, cycloalkyl groups are fully saturated.Examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl,cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, andcyclooctyl. Bicyclic cycloalkyl ring systems are bridged monocyclicrings or fused bicyclic rings. In embodiments, bridged monocyclic ringscontain a monocyclic cycloalkyl ring where two non adjacent carbon atomsof the monocyclic ring are linked by an alkylene bridge of between oneand three additional carbon atoms (i.e., a bridging group of the form(CH₂)_(w), where w is 1, 2, or 3). Representative examples of bicyclicring systems include, but are not limited to, bicyclo[3.1.1]heptane,bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, bicyclo[3.2.2]nonane,bicyclo[3.3.1]nonane, and bicyclo[4.2.1]nonane. In embodiments, fusedbicyclic cycloalkyl ring systems contain a monocyclic cycloalkyl ringfused to either a phenyl, a monocyclic cycloalkyl, a monocycliccycloalkenyl, a monocyclic heterocyclyl, or a monocyclic heteroaryl. Inembodiments, the bridged or fused bicyclic cycloalkyl is attached to theparent molecular moiety through any carbon atom contained within themonocyclic cycloalkyl ring. In embodiments, cycloalkyl groups areoptionally substituted with one or two groups which are independentlyoxo or thia. In embodiments, the fused bicyclic cycloalkyl is a 5 or 6membered monocyclic cycloalkyl ring fused to either a phenyl ring, a 5or 6 membered monocyclic cycloalkyl, a 5 or 6 membered monocycliccycloalkenyl, a 5 or 6 membered monocyclic heterocyclyl, or a 5 or 6membered monocyclic heteroaryl, wherein the fused bicyclic cycloalkyl isoptionally substituted by one or two groups which are independently oxoor thia. In embodiments, multicyclic cycloalkyl ring systems are amonocyclic cycloalkyl ring (base ring) fused to either (i) one ringsystem selected from the group consisting of a bicyclic aryl, a bicyclicheteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and abicyclic heterocyclyl; or (ii) two other ring systems independentlyselected from the group consisting of a phenyl, a bicyclic aryl, amonocyclic or bicyclic heteroaryl, a monocyclic or bicyclic cycloalkyl,a monocyclic or bicyclic cycloalkenyl, and a monocyclic or bicyclicheterocyclyl. In embodiments, the multicyclic cycloalkyl is attached tothe parent molecular moiety through any carbon atom contained within thebase ring. In embodiments, multicyclic cycloalkyl ring systems are amonocyclic cycloalkyl ring (base ring) fused to either (i) one ringsystem selected from the group consisting of a bicyclic aryl, a bicyclicheteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and abicyclic heterocyclyl; or (ii) two other ring systems independentlyselected from the group consisting of a phenyl, a monocyclic heteroaryl,a monocyclic cycloalkyl, a monocyclic cycloalkenyl, and a monocyclicheterocyclyl. Examples of multicyclic cycloalkyl groups include, but arenot limited to tetradecahydrophenanthrenyl, perhydrophenothiazin-1-yl,and perhydrophenoxazin-1-yl.

In embodiments, a cycloalkyl is a cycloalkenyl. The term “cycloalkenyl”is used in accordance with its plain ordinary meaning. In embodiments, acycloalkenyl is a monocyclic, bicyclic, or a multicyclic cycloalkenylring system. In embodiments, monocyclic cycloalkenyl ring systems arecyclic hydrocarbon groups containing from 3 to 8 carbon atoms, wheresuch groups are unsaturated (i.e., containing at least one annularcarbon carbon double bond), but not aromatic. Examples of monocycliccycloalkenyl ring systems include cyclopentenyl and cyclohexenyl. Inembodiments, bicyclic cycloalkenyl rings are bridged monocyclic rings ora fused bicyclic rings. In embodiments, bridged monocyclic rings containa monocyclic cycloalkenyl ring where two non adjacent carbon atoms ofthe monocyclic ring are linked by an alkylene bridge of between one andthree additional carbon atoms (i.e., a bridging group of the form(CH₂)_(w), where w is 1, 2, or 3). Representative examples of bicycliccycloalkenyls include, but are not limited to, norbornenyl andbicyclo[2.2.2]oct 2 enyl. In embodiments, fused bicyclic cycloalkenylring systems contain a monocyclic cycloalkenyl ring fused to either aphenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, a monocyclicheterocyclyl, or a monocyclic heteroaryl. In embodiments, the bridged orfused bicyclic cycloalkenyl is attached to the parent molecular moietythrough any carbon atom contained within the monocyclic cycloalkenylring. In embodiments, cycloalkenyl groups are optionally substitutedwith one or two groups which are independently oxo or thia. Inembodiments, multicyclic cycloalkenyl rings contain a monocycliccycloalkenyl ring (base ring) fused to either (i) one ring systemselected from the group consisting of a bicyclic aryl, a bicyclicheteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and abicyclic heterocyclyl; or (ii) two ring systems independently selectedfrom the group consisting of a phenyl, a bicyclic aryl, a monocyclic orbicyclic heteroaryl, a monocyclic or bicyclic cycloalkyl, a monocyclicor bicyclic cycloalkenyl, and a monocyclic or bicyclic heterocyclyl. Inembodiments, the multicyclic cycloalkenyl is attached to the parentmolecular moiety through any carbon atom contained within the base ring.In embodiments, multicyclic cycloalkenyl rings contain a monocycliccycloalkenyl ring (base ring) fused to either (i) one ring systemselected from the group consisting of a bicyclic aryl, a bicyclicheteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and abicyclic heterocyclyl; or (ii) two ring systems independently selectedfrom the group consisting of a phenyl, a monocyclic heteroaryl, amonocyclic cycloalkyl, a monocyclic cycloalkenyl, and a monocyclicheterocyclyl.

In embodiments, a heterocycloalkyl is a heterocyclyl. The term“heterocyclyl” as used herein, means a monocyclic, bicyclic, ormulticyclic heterocycle. The heterocyclyl monocyclic heterocycle is a 3,4, 5, 6 or 7 membered ring containing at least one heteroatomindependently selected from the group consisting of O, N, and S wherethe ring is saturated or unsaturated, but not aromatic. The 3 or 4membered ring contains 1 heteroatom selected from the group consistingof O, N and S. The 5 membered ring can contain zero or one double bondand one, two or three heteroatoms selected from the group consisting ofO, N and S. The 6 or 7 membered ring contains zero, one or two doublebonds and one, two or three heteroatoms selected from the groupconsisting of O, N and S. The heterocyclyl monocyclic heterocycle isconnected to the parent molecular moiety through any carbon atom or anynitrogen atom contained within the heterocyclyl monocyclic heterocycle.Representative examples of heterocyclyl monocyclic heterocycles include,but are not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl,1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl,imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl,isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl,oxadiazolidinyl, oxazolinyl, oxazolidinyl, piperazinyl, piperidinyl,pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl,tetrahydrofuranyl, tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl,thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxidothiomorpholinyl(thiomorpholine sulfone), thiopyranyl, and trithianyl. The heterocyclylbicyclic heterocycle is a monocyclic heterocycle fused to either aphenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, a monocyclicheterocycle, or a monocyclic heteroaryl. The heterocyclyl bicyclicheterocycle is connected to the parent molecular moiety through anycarbon atom or any nitrogen atom contained within the monocyclicheterocycle portion of the bicyclic ring system. Representative examplesof bicyclic heterocyclyls include, but are not limited to,2,3-dihydrobenzofuran-2-yl, 2,3-dihydrobenzofuran-3-yl, indolin-1-yl,indolin-2-yl, indolin-3-yl, 2,3-dihydrobenzothien-2-yl,decahydroquinolinyl, decahydroisoquinolinyl, octahydro-1H-indolyl, andoctahydrobenzofuranyl. In embodiments, heterocyclyl groups areoptionally substituted with one or two groups which are independentlyoxo or thia. In certain embodiments, the bicyclic heterocyclyl is a 5 or6 membered monocyclic heterocyclyl ring fused to a phenyl ring, a 5 or 6membered monocyclic cycloalkyl, a 5 or 6 membered monocycliccycloalkenyl, a 5 or 6 membered monocyclic heterocyclyl, or a 5 or 6membered monocyclic heteroaryl, wherein the bicyclic heterocyclyl isoptionally substituted by one or two groups which are independently oxoor thia. Multicyclic heterocyclyl ring systems are a monocyclicheterocyclyl ring (base ring) fused to either (i) one ring systemselected from the group consisting of a bicyclic aryl, a bicyclicheteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and abicyclic heterocyclyl; or (ii) two other ring systems independentlyselected from the group consisting of a phenyl, a bicyclic aryl, amonocyclic or bicyclic heteroaryl, a monocyclic or bicyclic cycloalkyl,a monocyclic or bicyclic cycloalkenyl, and a monocyclic or bicyclicheterocyclyl. The multicyclic heterocyclyl is attached to the parentmolecular moiety through any carbon atom or nitrogen atom containedwithin the base ring. In embodiments, multicyclic heterocyclyl ringsystems are a monocyclic heterocyclyl ring (base ring) fused to either(i) one ring system selected from the group consisting of a bicyclicaryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicycliccycloalkenyl, and a bicyclic heterocyclyl; or (ii) two other ringsystems independently selected from the group consisting of a phenyl, amonocyclic heteroaryl, a monocyclic cycloalkyl, a monocycliccycloalkenyl, and a monocyclic heterocyclyl. Examples of multicyclicheterocyclyl groups include, but are not limited to10H-phenothiazin-10-yl, 9,10-dihydroacridin-9-yl,9,10-dihydroacridin-10-yl, 10H-phenoxazin-10-yl,10,11-dihydro-5H-dibenzo[b,f]azepin-5-yl,1,2,3,4-tetrahydropyrido[4,3-g]isoquinolin-2-yl,12H-benzo[b]phenoxazin-12-yl, and dodecahydro-1H-carbazol-9-yl.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” includes, but is not limited to, fluoromethyl,difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl,3-bromopropyl, and the like.

The term “acyl” means, unless otherwise stated, —C(O)R where R is asubstituted or unsubstituted alkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent, which can be a single ring ormultiple rings (preferably from 1 to 3 rings) that are fused together(i.e., a fused ring aryl) or linked covalently. A fused ring aryl refersto multiple rings fused together wherein at least one of the fused ringsis an aryl ring. The term “heteroaryl” refers to aryl groups (or rings)that contain at least one heteroatom such as N, O, or S, wherein thenitrogen and sulfur atoms are optionally oxidized, and the nitrogenatom(s) are optionally quaternized. Thus, the term “heteroaryl” includesfused ring heteroaryl groups (i.e., multiple rings fused togetherwherein at least one of the fused rings is a heteroaromatic ring). A5,6-fused ring heteroarylene refers to two rings fused together, whereinone ring has 5 members and the other ring has 6 members, and wherein atleast one ring is a heteroaryl ring. Likewise, a 6,6-fused ringheteroarylene refers to two rings fused together, wherein one ring has 6members and the other ring has 6 members, and wherein at least one ringis a heteroaryl ring. And a 6,5-fused ring heteroarylene refers to tworings fused together, wherein one ring has 6 members and the other ringhas 5 members, and wherein at least one ring is a heteroaryl ring. Aheteroaryl group can be attached to the remainder of the moleculethrough a carbon or heteroatom. Non-limiting examples of aryl andheteroaryl groups include phenyl, naphthyl, pyrrolyl, pyrazolyl,pyridazinyl, triazinyl, pyrimidinyl, imidazolyl, pyrazinyl, purinyl,oxazolyl, isoxazolyl, thiazolyl, furyl, thienyl, pyridyl, pyrimidyl,benzothiazolyl, benzoxazoyl benzimidazolyl, benzofuran, isobenzofuranyl,indolyl, isoindolyl, benzothiophenyl, isoquinolyl, quinoxalinyl,quinolyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl,3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl,2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl,4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl,2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl,5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Substituents for each of the above notedaryl and heteroaryl ring systems are selected from the group ofacceptable substituents described below. An “arylene” and a“heteroarylene,” alone or as part of another substituent, mean adivalent radical derived from an aryl and heteroaryl, respectively. Aheteroaryl group substituent may be —O— bonded to a ring heteroatomnitrogen.

Spirocyclic rings are two or more rings wherein adjacent rings areattached through a single atom. The individual rings within spirocyclicrings may be identical or different. Individual rings in spirocyclicrings may be substituted or unsubstituted and may have differentsubstituents from other individual rings within a set of spirocyclicrings. Possible substituents for individual rings within spirocyclicrings are the possible substituents for the same ring when not part ofspirocyclic rings (e.g. substituents for cycloalkyl or heterocycloalkylrings). Spirocylic rings may be substituted or unsubstituted cycloalkyl,substituted or unsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkyl or substituted or unsubstituted heterocycloalkylene andindividual rings within a spirocyclic ring group may be any of theimmediately previous list, including having all rings of one type (e.g.all rings being substituted heterocycloalkylene wherein each ring may bethe same or different substituted heterocycloalkylene). When referringto a spirocyclic ring system, heterocyclic spirocyclic rings means aspirocyclic rings wherein at least one ring is a heterocyclic ring andwherein each ring may be a different ring. When referring to aspirocyclic ring system, substituted spirocyclic rings means that atleast one ring is substituted and each substituent may optionally bedifferent.

The symbol “

” denotes the point of attachment of a chemical moiety to the remainderof a molecule or chemical formula.

The term “oxo,” as used herein, means an oxygen that is double bonded toa carbon atom.

The term “alkylarylene” as an arylene moiety covalently bonded to analkylene moiety (also referred to herein as an alkylene linker). Inembodiments, the alkylarylene group has the formula:

An alkylarylene moiety may be substituted (e.g. with a substituentgroup) on the alkylene moiety or the arylene linker (e.g. at carbons 2,3, 4, or 6) with halogen, oxo, —N₃, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN, —CHO,—OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₂CH₃—SO₃H, —OSO₃H, —SO₂NH₂,—NHNH₂, —ONH₂, —NHC(O)NHNH₂, substituted or unsubstituted C₁-C₅ alkyl orsubstituted or unsubstituted 2 to 5 membered heteroalkyl). Inembodiments, the alkylarylene is unsubstituted.

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “cycloalkyl,”“heterocycloalkyl,” “aryl,” and “heteroaryl”) includes both substitutedand unsubstituted forms of the indicated radical. Preferred substituentsfor each type of radical are provided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —NR′NR″R′″,—ONR′R″, —NR′C(O)NR″NR′″ R″″, —CN, —NO₂, —NR′SO₂R″, —NR′C(O)R″,—NR′C(O)—OR″, —NR′OR″, in a number ranging from zero to (2m′+1), wherem′ is the total number of carbon atoms in such radical. R, R′, R″, R′″,and R″″ each preferably independently refer to hydrogen, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl (e.g., aryl substituted with 1-3 halogens),substituted or unsubstituted heteroaryl, substituted or unsubstitutedalkyl, alkoxy, or thioalkoxy groups, or arylalkyl groups. When acompound described herein includes more than one R group, for example,each of the R groups is independently selected as are each R′, R″, R′″,and R″″ group when more than one of these groups is present. When R′ andR″ are attached to the same nitrogen atom, they can be combined with thenitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For example,—NR′R″ includes, but is not limited to, 1-pyrrolidinyl and4-morpholinyl. From the above discussion of substituents, one of skillin the art will understand that the term “alkyl” is meant to includegroups including carbon atoms bound to groups other than hydrogengroups, such as haloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl (e.g.,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are varied and areselected from, for example: —OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″,—OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′,—NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″,—S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —NR′NR″R′″, —ONR′R″,—NR′C(O)NR″NR′″ R″″, —CN, —NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy,and fluoro(C₁-C₄)alkyl, —NR′SO₂R″, —NR′C(O)R″, —NR′C(O)—OR″, —NR′OR″, ina number ranging from zero to the total number of open valences on thearomatic ring system; and where R′, R″, R′″, and R″″ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl. When a compound described herein includes more than one Rgroup, for example, each of the R groups is independently selected asare each R′, R″, R′″, and R″″ groups when more than one of these groupsis present.

Substituents for rings (e.g. cycloalkyl, heterocycloalkyl, aryl,heteroaryl, cycloalkylene, heterocycloalkylene, arylene, orheteroarylene) may be depicted as substituents on the ring rather thanon a specific atom of a ring (commonly referred to as a floatingsubstituent). In such a case, the substituent may be attached to any ofthe ring atoms (obeying the rules of chemical valency) and in the caseof fused rings or spirocyclic rings, a substituent depicted asassociated with one member of the fused rings or spirocyclic rings (afloating substituent on a single ring), may be a substituent on any ofthe fused rings or spirocyclic rings (a floating substituent on multiplerings). When a substituent is attached to a ring, but not a specificatom (a floating substituent), and a subscript for the substituent is aninteger greater than one, the multiple substituents may be on the sameatom, same ring, different atoms, different fused rings, differentspirocyclic rings, and each substituent may optionally be different.Where a point of attachment of a ring to the remainder of a molecule isnot limited to a single atom (a floating substituent), the attachmentpoint may be any atom of the ring and in the case of a fused ring orspirocyclic ring, any atom of any of the fused rings or spirocyclicrings while obeying the rules of chemical valency. Where a ring, fusedrings, or spirocyclic rings contain one or more ring heteroatoms and thering, fused rings, or spirocyclic rings are shown with one more floatingsubstituents (including, but not limited to, points of attachment to theremainder of the molecule), the floating substituents may be bonded tothe heteroatoms. Where the ring heteroatoms are shown bound to one ormore hydrogens (e.g. a ring nitrogen with two bonds to ring atoms and athird bond to a hydrogen) in the structure or formula with the floatingsubstituent, when the heteroatom is bonded to the floating substituent,the substituent will be understood to replace the hydrogen, whileobeying the rules of chemical valency.

Two or more substituents may optionally be joined to form aryl,heteroaryl, cycloalkyl, or heterocycloalkyl groups. Such so-calledring-forming substituents are typically, though not necessarily, foundattached to a cyclic base structure. In one embodiment, the ring-formingsubstituents are attached to adjacent members of the base structure. Forexample, two ring-forming substituents attached to adjacent members of acyclic base structure create a fused ring structure. In anotherembodiment, the ring-forming substituents are attached to a singlemember of the base structure. For example, two ring-forming substituentsattached to a single member of a cyclic base structure create aspirocyclic structure. In yet another embodiment, the ring-formingsubstituents are attached to non-adjacent members of the base structure.

As used herein, the terms “heteroatom” or “ring heteroatom” are meant toinclude oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), andsilicon (Si).

The term “non-covalent linker” is used in accordance with its ordinarymeaning and refers to a divalent moiety which includes at least twomolecules that are not covalently linked to each other but are capableof interacting with each other via a non-covalent bond (e.g.electrostatic interactions (e.g. ionic bond, hydrogen bond, halogenbond) or van der Waals interactions (e.g. dipole-dipole, dipole-induceddipole, London dispersion). In embodiments, the non-covalent linker isthe result of two molecules that are not covalently linked to each otherthat interact with each other via a non-covalent bond.

The term “anchor moiety” as used herein refers to a chemical moietycapable of interacting (e.g., covalently or non-covalently) with asecond, optionally different, chemical moiety (e.g., complementaryanchor moiety binder). In embodiments, the anchor moiety is abioconjugate reactive group capable of interacting (e.g., covalently)with a complementary bioconjugate reactive group (e.g., complementaryanchor moiety reactive group). In embodiments, an anchor moiety is aclick chemistry reactant moiety. In embodiments, the anchor moiety (an“affinity anchor moiety”) is capable of non-covalently interacting witha second chemical moiety (e.g., complementary affinity anchor moietybinder). Non-limiting examples of an anchor moiety include biotin,azide, trans-cyclooctene (TCO) and phenyl boric acid (PBA). Inembodiments, an affinity anchor moiety (e.g., biotin moiety) interactsnon-covalently with a complementary affinity anchor moiety binder (e.g.,streptavidin moiety). In embodiments, an anchor moiety (e.g., azidemoiety, trans-cyclooctene (TCO) moiety, phenyl boric acid (PBA) moiety)covalently binds a complementary anchor moiety binder (e.g.,dibenzocyclooctyne (DBCO) moiety, tetrazine (TZ) moiety,salicylhydroxamic acid (SHA) moiety).

The terms “cleavable linker” or “cleavable moiety” as used herein refersto a divalent or monovalent, respectively, moiety which is capable ofbeing separated (e.g., detached, split, disconnected, hydrolyzed, astable bond within the moiety is broken) into distinct entities. Acleavable linker is cleavable (e.g., specifically cleavable) in responseto external stimuli (e.g., enzymes, nucleophilic/basic reagents,reducing agents, photo-irradiation, electrophilic/acidic reagents,organometallic and metal reagents, or oxidizing reagents). A chemicallycleavable linker refers to a linker which is capable of being split inresponse to the presence of a chemical (e.g., acid, base, oxidizingagent, reducing agent, Pd(0), tris-(2-carboxyethyl)phosphine, dilutenitrous acid, fluoride, tris(3-hydroxypropyl)phosphine), sodiumdithionite (Na₂S₂O₄), hydrazine (N₂H₄)). A chemically cleavable linkeris non-enzymatically cleavable. In embodiments, the cleavable linker iscleaved by contacting the cleavable linker with a cleaving agent. Inembodiments, the cleaving agent is sodium dithionite (Na₂S₂O₄), weakacid, hydrazine (N₂H₄), Pd(0), or light-irradiation (e.g., ultravioletradiation).

A photocleavable linker (e.g., including or consisting of ao-nitrobenzyl group) refers to a linker which is capable of being splitin response to photo-irradiation (e.g., ultraviolet radiation). Anacid-cleavable linker refers to a linker which is capable of being splitin response to a change in the pH (e.g., increased acidity). Abase-cleavable linker refers to a linker which is capable of being splitin response to a change in the pH (e.g., decreased acidity). Anoxidant-cleavable linker refers to a linker which is capable of beingsplit in response to the presence of an oxidizing agent. Areductant-cleavable linker refers to a linker which is capable of beingsplit in response to the presence of an reducing agent (e.g.,Tris(3-hydroxypropyl)phosphine). In embodiments, the cleavable linker isa dialkylketal linker, an azo linker, an allyl linker, a cyanoethyllinker, a 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl linker, or anitrobenzyl linker.

The term “orthogonally cleavable linker” or “orthogonal cleavablelinker” as used herein refer to a cleavable linker that is cleaved by afirst cleaving agent (e.g., enzyme, nucleophilic/basic reagent, reducingagent, photo-irradiation, electrophilic/acidic reagent, organometallicand metal reagent, oxidizing reagent) in a mixture of two or moredifferent cleaving agents and is not cleaved by any other differentcleaving agent in the mixture of two or more cleaving agents. Forexample, two different cleavable linkers are both orthogonal cleavablelinkers when a mixture of the two different cleavable linkers arereacted with two different cleaving agents and each cleavable linker iscleaved by only one of the cleaving agents and not the other cleavingagent. In embodiments, an orthogonally is a cleavable linker thatfollowing cleavage the two separated entities (e.g., fluorescent dye,bioconjugate reactive group) do not further react and form a neworthogonally cleavable linker.

The term “orthogonal binding group” or “orthogonal binding molecule” asused herein refer to a binding group (e.g. anchor moiety orcomplementary anchor moiety binder) that is capable of binding a firstcomplementary binding group (e.g., complementary anchor moiety binder oranchor moiety) in a mixture of two or more different complementarybinding groups and is unable to bind any other different complementarybinding group in the mixture of two or more complementary bindinggroups. For example, two different binding groups are both orthogonalbinding groups when a mixture of the two different binding groups arereacted with two complementary binding groups and each binding groupbinds only one of the complementary binding groups and not the othercomplementary binding group. An example of a set of four orthogonalbinding groups and a set of orthogonal complementary binding groups arethe binding groups biotin, azide, trans-cyclooctene (TCO) and phenylboric acid (PBA), which specifically and efficiently bind or react withthe complementary binding groups streptavidin, dibenzocyclooctyne(DBCO), tetrazine (TZ) and salicylhydroxamic acid (SHA) respectively.

The term “orthogonal detectable label” or “orthogonal detectable moiety”as used herein refer to a detectable label (e.g. fluorescent dye ordetectable dye) that is capable of being detected and identified (e.g.,by use of a detection means (e.g., emission wavelength, physicalcharacteristic measurement)) in a mixture or a panel (collection ofseparate samples) of two or more different detectable labels. Forexample, two different detectable labels that are fluorescent dyes areboth orthogonal detectable labels when a panel of the two differentfluorescent dyes is subjected to a wavelength of light that is absorbedby one fluorescent dye but not the other and results in emission oflight from the fluorescent dye that absorbed the light but not the otherfluorescent dye. Orthogonal detectable labels may be separatelyidentified by different absorbance or emission intensities of theorthogonal detectable labels compared to each other and not only be theabsolute presence of absence of a signal. An example of a set of fourorthogonal detectable labels is the set of Rox-Labeled Tetrazine,Alexa488-Labeled SHA, Cy5-Labeled Streptavidin, and R6G-LabeledDibenzocyclooctyne.

The term “polymerase-compatible cleavable moiety” as used herein refersa cleavable moiety which does not interfere with the function of apolymerase (e.g., DNA polymerase, modified DNA polymerase). Methods fordetermining the function of a polymerase contemplated herein aredescribed in B. Rosenblum et al. (Nucleic Acids Res. 1997 Nov. 15;25(22): 4500-4504); and Z. Zhu et al. (Nucleic Acids Res. 1994 Aug. 25;22(16): 3418-3422), which are incorporated by reference herein in theirentirety for all purposes. In embodiments the polymerase-compatiblecleavable moiety does not decrease the function of a polymerase relativeto the absence of the polymerase-compatible cleavable moiety. Inembodiments, the polymerase-compatible cleavable moiety does notnegatively affect DNA polymerase recognition. In embodiments, thepolymerase-compatible cleavable moiety does not negatively affect (e.g.,limit) the read length of the DNA polymerase. Additional examples of apolymerase-compatible cleavable moiety may be found in U.S. Pat. No.6,664,079, Ju J. et al. (2006) Proc Natl Acad Sci USA103(52):19635-19640; Ruparel H. et al. (2005) Proc Natl Acad Sci USA102(17):5932-5937; Wu J. et al. (2007) Proc Natl Acad Sci USA104(104):16462-16467; Guo J. et al. (2008) Proc Natl Acad Sci USA105(27): 9145-9150 Bentley D. R. et al. (2008) Nature 456(7218):53-59;or Hutter D. et al. (2010) Nucleosides Nucleotides & Nucleic Acids29:879-895, which are incorporated herein by reference in their entiretyfor all purposes. In embodiments, a polymerase-compatible cleavablemoiety includes an azido moiety or a dithiol linking moiety. Inembodiments, the polymerase-compatible cleavable moiety is —NH₂, —CN,—CH₃, C₂-C₆ allyl (e.g., —CH₂—CH═CH₂), methoxyalkyl (e.g., —CH₂—O—CH₃),or —CH₂N₃. In embodiments, the polymerase-compatible cleavable moietycomprises a disulfide moiety.

The term “allyl” as described herein refers to an unsubstitutedmethylene attached to a vinyl group (i.e. —CH═CH₂), having the formula

An “allyl linker” refers to a divalent unsubstituted methylene attachedto a vinyl group, having the formula

A “detectable agent” or “detectable compound” or “detectable label” or“detectable moiety” is a composition detectable by spectroscopic,photochemical, biochemical, immunochemical, chemical, magnetic resonanceimaging, or other physical means. For example, detectable agents include¹⁸F, ³²P, ³³P, ⁴⁵Ti, ⁴⁷Sc, ⁵²Fe, ⁵⁹Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga,⁷⁷As, ⁸⁶Y, ⁹⁰Y, ⁸⁹Sr, ⁸⁹Zr, ⁹⁴Tc, ⁹⁴Tc, ^(99m)Tc, ⁹⁹Mo, ¹⁰⁵Pd, ¹⁰⁵Rh,¹¹¹Ag, ¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁴²Pr, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁵³Sm,¹⁵⁴⁻¹⁵⁸¹Gd, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁶⁹Er, ¹⁷⁵Lu, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re,¹⁸⁹Re, ¹⁹⁴F, ¹⁹⁸Au, ¹⁹⁹Au, ²¹¹At, ²¹¹Pb, ²¹²Bi, ²¹²Pb, ²¹³Bi, ²²³Ra,²²⁵Ac, Cr, V, Mn, Fe, Co, Ni, Cu, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb,Dy, Ho, Er, Tm, Yb, Lu, ³²P, fluorophore (e.g. fluorescent dyes),modified oligonucleotides (e.g., moieties described inPCT/US2015/022063, which is incorporated herein by reference),electron-dense reagents, enzymes (e.g., as commonly used in an ELISA),biotin, digoxigenin, paramagnetic molecules, paramagnetic nanoparticles,ultrasmall superparamagnetic iron oxide (“USPIO”) nanoparticles, USPIOnanoparticle aggregates, superparamagnetic iron oxide (“SPIO”)nanoparticles, SPIO nanoparticle aggregates, monochrystalline iron oxidenanoparticles, monochrystalline iron oxide, nanoparticle contrastagents, liposomes or other delivery vehicles containing Gadoliniumchelate (“Gd-chelate”) molecules, Gadolinium, radioisotopes,radionuclides (e.g. carbon-11, nitrogen-13, oxygen-15, fluorine-18,rubidium-82), fluorodeoxyglucose (e.g. fluorine-18 labeled), any gammaray emitting radionuclides, positron-emitting radionuclide, radiolabeledglucose, radiolabeled water, radiolabeled ammonia, biocolloids,microbubbles (e.g. including microbubble shells including albumin,galactose, lipid, and/or polymers; microbubble gas core including air,heavy gas(es), perfluorcarbon, nitrogen, octafluoropropane, perflexanelipid microsphere, perflutren, etc.), iodinated contrast agents (e.g.iohexol, iodixanol, ioversol, iopamidol, ioxilan, iopromide,diatrizoate, metrizoate, ioxaglate), barium sulfate, thorium dioxide,gold, gold nanoparticles, gold nanoparticle aggregates, fluorophores,two-photon fluorophores, or haptens and proteins or other entities whichcan be made detectable, e.g., by incorporating a radiolabel into apeptide or antibody specifically reactive with a target peptide.

Radioactive substances (e.g., radioisotopes) that may be used as imagingand/or labeling agents in accordance with the embodiments of thedisclosure include, but are not limited to, ¹⁸F, ³²P, ³³P, ⁴⁵Ti, ⁴⁷Sc,⁵²Fe, ⁵⁹Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁷As, ⁸⁶Y, ⁹⁰Y, ⁸⁹Sr, ⁸⁹Zr,⁹⁴Tc, ⁹⁴Tc, ^(99m)Tc, ⁹⁹Mo, ¹⁰⁵Pd, ¹⁰⁵Rh, ¹¹¹Ag, ¹¹¹In, ¹²³I, ¹²⁴I,¹²⁵I, ¹³¹I, ¹⁴²Pr, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁵⁴⁻¹⁵⁸¹Gd, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho,¹⁶⁹Er, ¹⁷⁵Lu, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ¹⁹⁴Ir, ¹⁹⁸Au, ¹⁹⁹Au, ²¹¹At,²¹¹Pb, ²¹²Bi, ²¹²Pb, ²¹³Bi, ²²³Ra and ²²⁵Ac. Paramagnetic ions that maybe used as additional imaging agents in accordance with the embodimentsof the disclosure include, but are not limited to, ions of transitionand lanthanide metals (e.g. metals having atomic numbers of 21-29, 42,43, 44, or 57-71). These metals include ions of Cr, V, Mn, Fe, Co, Ni,Cu, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu.

Examples of detectable agents include imaging agents, includingfluorescent and luminescent substances, including, but not limited to, avariety of organic or inorganic small molecules commonly referred to as“dyes,” “labels,” or “indicators.” Examples include fluorescein,rhodamine, acridine dyes, Alexa dyes, and cyanine dyes. In embodiments,the detectable moiety is a fluorescent molecule (e.g., acridine dye,cyanine, dye, fluorine dye, oxazine dye, phenanthridine dye, orrhodamine dye). In embodiments, the detectable moiety is a fluorescentmolecule (e.g., acridine dye, cyanine, dye, fluorine dye, oxazine dye,phenanthridine dye, or rhodamine dye). In embodiments, the detectablemoiety is a fluorescein isothiocyanate moiety,tetramethylrhodamine-5-(and 6)-isothiocyanate moiety, Cy2 moeity, Cy3moiety, Cy5 moiety, Cy7 moiety, 4′,6-diamidino-2-phenylindole moiety,Hoechst 33258 moiety, Hoechst 33342 moiety, Hoechst 34580 moiety,propidium-iodide moiety, or acridine orange moiety. In embodiments, thedetectable moiety is a Indo-1, Ca saturated moiety, Indo-1 Ca2+ moiety,Cascade Blue BSA pH 7.0 moiety, Cascade Blue moiety, LysoTracker Bluemoiety, Alexa 405 moiety, LysoSensor Blue pH 5.0 moiety, LysoSensor Bluemoiety, DyLight 405 moiety, DyLight 350 moiety, BFP (Blue FluorescentProtein) moiety, Alexa 350 moiety, 7-Amino-4-methylcoumarin pH 7.0moiety, Amino Coumarin moiety, AMCA conjugate moiety, Coumarin moiety,7-Hydroxy-4-methylcoumarin moiety, 7-Hydroxy-4-methylcoumarin pH 9.0moiety, 6,8-Difluoro-7-hydroxy-4-methylcoumarin pH 9.0 moiety, Hoechst33342 moiety, Pacific Blue moiety, Hoechst 33258 moiety, Hoechst33258-DNA moiety, Pacific Blue antibody conjugate pH 8.0 moiety,PO-PRO-1 moiety, PO-PRO-1-DNA moiety, POPO-1 moiety, POPO-1-DNA moiety,DAPI-DNA moiety, DAPI moiety, Marina Blue moiety, SYTOX Blue-DNA moiety,CFP (Cyan Fluorescent Protein) moiety, eCFP (Enhanced Cyan FluorescentProtein) moiety, 1-Anilinonaphthalene-8-sulfonic acid (1,8-ANS) moiety,Indo-1, Ca free moiety, 1,8-ANS (1-Anilinonaphthalene-8-sulfonic acid)moiety, BO-PRO-1-DNA moiety, BOPRO-1 moiety, BOBO-1-DNA moiety, SYTO45-DNA moiety, evoglow-Pp1 moiety, evoglow-Bs1 moiety, evoglow-Bs2moiety, Auramine O moiety, DiO moiety, LysoSensor Green pH 5.0 moiety,Cy 2 moiety, LysoSensor Green moiety, Fura-2, high Ca moiety, Fura-2Ca2+sup> moiety, SYTO 13-DNA moiety, YO-PRO-1-DNA moiety, YOYO-1-DNAmoiety, eGFP (Enhanced Green Fluorescent Protein) moiety, LysoTrackerGreen moiety, GFP (S65T) moiety, BODIPY FL, MeOH moiety, Sapphiremoiety, BODIPY FL conjugate moiety, MitoTracker Green moiety,MitoTracker Green FM, MeOH moiety, Fluorescein 0.1 M NaOH moiety,Calcein pH 9.0 moiety, Fluorescein pH 9.0 moiety, Calcein moiety,Fura-2, no Ca moiety, Fluo-4 moiety, FDA moiety, DTAF moiety,Fluorescein moiety, CFDA moiety, FITC moiety, Alexa Fluor 488hydrazide-water moiety, DyLight 488 moiety, 5-FAM pH 9.0 moiety, Alexa488 moiety, Rhodamine 110 moiety, Rhodamine 110 pH 7.0 moiety, AcridineOrange moiety, BCECF pH 5.5 moiety, PicoGreendsDNA quantitation reagentmoiety, SYBR Green I moiety, Rhodaminen Green pH 7.0 moiety, CyQUANTGR-DNA moiety, NeuroTrace 500/525, green fluorescent Nissl stain-RNAmoiety, DansylCadaverine moiety, Fluoro-Emerald moiety, Nissl moiety,Fluorescein dextran pH 8.0 moiety, Rhodamine Green moiety,5-(and-6)-Carboxy-2′, 7′-dichlorofluorescein pH 9.0 moiety,DansylCadaverine, MeOH moiety, eYFP (Enhanced Yellow FluorescentProtein) moiety, Oregon Green 488 moiety, Fluo-3 moiety, BCECF pH 9.0moiety, SBFI-Na+ moiety, Fluo-3 Ca2+ moiety, Rhodamine 123 MeOH moiety,FlAsH moiety, Calcium Green-1 Ca2+ moiety, Magnesium Green moiety,DM-NERF pH 4.0 moiety, Calcium Green moiety, Citrine moiety, LysoSensorYellow pH 9.0 moiety, TO-PRO-1-DNA moiety, Magnesium Green Mg2+ moiety,Sodium Green Na+ moiety, TOTO-1-DNA moiety, Oregon Green 514 moiety,Oregon Green 514 antibody conjugate pH 8.0 moiety, NBD-X moiety, DM-NERFpH 7.0 moiety, NBD-X, MeOH moiety, CI-NERF pH 6.0 moiety, Alexa 430moiety, CI-NERF pH 2.5 moiety, Lucifer Yellow, CH moiety, LysoSensorYellow pH 3.0 moiety, 6-TET, SE pH 9.0 moiety, Eosin antibody conjugatepH 8.0 moiety, Eosin moiety, 6-Carboxyrhodamine 6G pH 7.0 moiety,6-Carboxyrhodamine 6G, hydrochloride moiety, Bodipy R6G SE moiety,BODIPY R6G MeOH moiety, 6 JOE moiety, Cascade Yellow moiety, mBananamoiety, Alexa 532 moiety, Erythrosin-5-isothiocyanate pH 9.0 moiety,6-HEX, SE pH 9.0 moiety, mOrange moiety, mHoneydew moiety, Cy 3 moiety,Rhodamine B moiety, DiI moiety, 5-TAMRA-MeOH moiety, Alexa 555 moiety,DyLight 549 moiety, BODIPY TMR-X, SE moiety, BODIPY TMR-X MeOH moiety,PO-PRO-3-DNA moiety, PO-PRO-3 moiety, Rhodamine moiety, POPO-3 moiety,Alexa 546 moiety, Calcium Orange Ca2+ moiety, TRITC moiety, CalciumOrange moiety, Rhodaminephalloidin pH 7.0 moiety, MitoTracker Orangemoiety, MitoTracker Orange MeOH moiety, Phycoerythrin moiety, MagnesiumOrange moiety, R-Phycoerythrin pH 7.5 moiety, 5-TAMRA pH 7.0 moiety,5-TAMRA moiety, Rhod-2 moiety, FM 1-43 moiety, Rhod-2 Ca2+ moiety, FM1-43 lipid moiety, LOLO-1-DNA moiety, dTomato moiety, DsRed moiety,Dapoxyl (2-aminoethyl) sulfonamide moiety, Tetramethylrhodamine dextranpH 7.0 moiety, Fluor-Ruby moiety, Resorufin moiety, Resorufin pH 9.0moiety, mTangerine moiety, LysoTracker Red moiety, Lissaminerhodaminemoiety, Cy 3.5 moiety, Rhodamine Red-X antibody conjugate pH 8.0 moiety,Sulforhodamine 101 EtOH moiety, JC-1 pH 8.2 moiety, JC-1 moiety,mStrawberry moiety, MitoTracker Red moiety, MitoTracker Red, MeOHmoiety, X-Rhod-1 Ca2+ moiety, Alexa 568 moiety, 5-ROX pH 7.0 moiety,5-ROX (5-Carboxy-X-rhodamine, triethylammonium salt) moiety,BO-PRO-3-DNA moiety, BOPRO-3 moiety, BOBO-3-DNA moiety, Ethidium Bromidemoiety, ReAsH moiety, Calcium Crimson moiety, Calcium Crimson Ca2+moiety, mRFP moiety, mCherry moiety, HcRed moiety, DyLight 594 moiety,Ethidium homodimer-1-DNA moiety, Ethidiumhomodimer moiety, PropidiumIodide moiety, SYPRO Ruby moiety, Propidium Iodide-DNA moiety, Alexa 594moiety, BODIPY TR-X, SE moiety, BODIPY TR-X, MeOH moiety, BODIPY TR-Xphallacidin pH 7.0 moiety, Alexa Fluor 610 R-phycoerythrin streptavidinpH 7.2 moiety, YO-PRO-3-DNA moiety, Di-8 ANEPPS moiety,Di-8-ANEPPS-lipid moiety, YOYO-3-DNA moiety, Nile Red-lipid moiety, NileRed moiety, DyLight 633 moiety, mPlum moiety, TO-PRO-3-DNA moiety, DDAOpH 9.0 moiety, Fura Red high Ca moiety, Allophycocyanin pH 7.5 moiety,APC (allophycocyanin) moiety, Nile Blue, EtOH moiety, TOTO-3-DNA moiety,Cy 5 moiety, BODIPY 650/665-X, MeOH moiety, Alexa Fluor 647R-phycoerythrin streptavidin pH 7.2 moiety, DyLight 649 moiety, Alexa647 moiety, Fura Red Ca2+ moiety, Atto 647 moiety, Fura Red, low Camoiety, Carboxynaphthofluorescein pH 10.0 moiety, Alexa 660 moiety, Cy5.5 moiety, Alexa 680 moiety, DyLight 680 moiety, Alexa 700 moiety, FM4-64, 2% CHAPS moiety, or FM 4-64 moiety. In embodiments, the dectablemoiety is a moiety of 1,1-Diethyl-4,4-carbocyanine iodide,1,2-Diphenylacetylene, 1,4-Diphenylbutadiene, 1,4-Diphenylbutadiyne,1,6-Diphenylhexatriene, 1,6-Diphenylhexatriene,1-anilinonaphthalene-8-sulfonic acid, 2,7-Dichlorofluorescein,2,5-DIPHENYLOXAZOLE, 2-Di-1-ASP, 2-dodecylresorufin,2-Methylbenzoxazole, 3,3-Diethylthiadicarbocyanine iodide,4-Dimethylamino-4-Nitrostilbene, 5(6)-Carboxyfluorescein,5(6)-Carboxynaphtofluorescein, 5(6)-Carboxytetramethylrhodamine B,5-(and-6)-carboxy-2′,7′-dichlorofluorescein,5-(and-6)-carboxy-2,7-dichlorofluorescein, 5-(N-hexadecanoyl)aminoeosin,5-(N-hexadecanoyl)aminoeosin, 5-chloromethylfluorescein, 5-FAM, 5-ROX,5-TAMRA, 5-TAMRA, 6,8-difluoro-7-hydroxy-4-methylcoumarin,6,8-difluoro-7-hydroxy-4-methylcoumarin, 6-carboxyrhodamine 6G, 6-HEX,6-JOE, 6-JOE, 6-TET, 7-aminoactinomycin D,7-Benzylamino-4-Nitrobenz-2-Oxa-1,3-Diazole, 7-Methoxycoumarin-4-AceticAcid, 8-Benzyloxy-5,7-diphenylquinoline,8-Benzyloxy-5,7-diphenylquinoline, 9,10-Bis(Phenylethynyl)Anthracene,9,10-Diphenylanthracene, 9-METHYLCARBAZOLE, (CS)2Ir(μ-Cl)2Ir(CS)2, AAA,Acridine Orange, Acridine Orange, Acridine Yellow, Acridine Yellow,Adams Apple Red 680, Adirondack Green 520, Alexa Fluor 350, Alexa Fluor405, Alexa Fluor 430, Alexa Fluor 430, Alexa Fluor 480, Alexa Fluor 488,Alexa Fluor 488, Alexa Fluor 488 hydrazide, Alexa Fluor 500, Alexa Fluor514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 546, Alexa Fluor 555,Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 594,Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 610-R-PE, Alexa Fluor 633,Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 647, Alexa Fluor 647-R-PE,Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 680-APC, Alexa Fluor680-R-PE, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790,Allophycocyanin, AmCyan1, Aminomethylcoumarin, Amplex Gold (product),Amplex Red Reagent, Amplex UltraRed, Anthracene, APC, APC-Seta-750,AsRed2, ATTO 390, ATTO 425, ATTO 430LS, ATTO 465, ATTO 488, ATTO 490LS,ATTO 495, ATTO 514, ATTO 520, ATTO 532, ATTO 550, ATTO 565, ATTO 590,ATTO 594, ATTO 610, ATTO 620, ATTO 633, ATTO 635, ATTO 647, ATTO 647N,ATTO 655, ATTO 665, ATTO 680, ATTO 700, ATTO 725, ATTO 740, ATTO Oxa12,ATTO Rho3B, ATTO Rho6G, ATTO Rho11, ATTO Rho12, ATTO Rho13, ATTO Rho14,ATTO Rho101, ATTO Thio12, Auramine O, Azami Green, Azami Greenmonomeric, B-phycoerythrin, BCECF, BCECF, Bex1, Biphenyl, Birch Yellow580, Blue-green algae, BO-PRO-1, BO-PRO-3, BOBO-1, BOBO-3, BODIPY 630650-X, BODIPY 650/665-X, BODIPY FL, BODIPY FL, BODIPY R6G, BODIPY TMR-X,BODIPY TR-X, BODIPY TR-X Ph 7.0, BODIPY TR-X phallacidin, BODIPY-DiMe,BODIPY-Phenyl, BODIPY-TMSCC, C3-Indocyanine, C3-Indocyanine,C3-Oxacyanine, C3-Thiacyanine Dye (EtOH), C3-Thiacyanine Dye (PrOH),C5-Indocyanine, C5-Oxacyanine, C5-Thiacyanine, C7-Indocyanine,C7-Oxacyanine, C545T, C-Phycocyanin, Calcein, Calcein red-orange,Calcium Crimson, Calcium Green-1, Calcium Orange, Calcofluor white 2MR,Carboxy SNARF-1 pH 6.0, Carboxy SNARF-1 pH 9.0,Carboxynaphthofluorescein, Cascade Blue, Cascade Yellow, Catskill Green540, CBQCA, CellMask Orange, CellTrace BODIPY TR methyl ester, CellTracecalcein violet, CellTrace™ Far Red, CellTracker Blue, CellTracker RedCMTPX, CellTracker Violet BMQC, CF405M, CF405S, CF488A, CF543, CF555,CFP, CFSE, CF™ 350, CF™ 485, Chlorophyll A, Chlorophyll B, Chromeo 488,Chromeo 494, Chromeo 505, Chromeo 546, Chromeo 642, Citrine, Citrine,ClOH butoxy aza-BODIPY, ClOH C12 aza-BODIPY, CM-H2DCFDA, Coumarin 1,Coumarin 6, Coumarin 6, Coumarin 30, Coumarin 314, Coumarin 334,Coumarin 343, Coumarine 545T, Cresyl Violet Perchlorate, CryptoLightCF1, CryptoLight CF2, CryptoLight CF3, CryptoLight CF4, CryptoLight CF5,CryptoLight CF6, Crystal Violet, Cumarin153, Cy2, Cy3, Cy3, Cy3.5, Cy3B,Cy3B, Cy3Cy5 ET, Cy5, Cy5, Cy5.5, Cy7, Cyanine3 NHS ester, Cyanine5carboxylic acid, Cyanine5 NHS ester, Cyclotella meneghiniana Kützing,CypHer5, CypHer5 pH 9.15, CyQUANT GR, CyTrak Orange, Dabcyl SE, DAF-FM,DAMC (Weiss), dansyl cadaverine, Dansyl Glycine (Dioxane), DAPI, DAPI,DAPI, DAPI, DAPI (DMSO), DAPI (H2O), Dapoxyl (2-aminoethyl)sulfonamide,DCI, DCM, DCM, DCM (acetonitrile), DCM (MeOH), DDAO, Deep Purple,di-8-ANEPPS, DiA, Dichlorotris(1,10-phenanthroline) ruthenium(II),DiClOH C12 aza-BODIPY, DiClOHbutoxy aza-BODIPY, DiD, DiI, DiIC18(3),DiO, DiR, Diversa Cyan-FP, Diversa Green-FP, DM-NERF pH 4.0, DOCI,Doxorubicin, DPP pH-Probe 590-7.5, DPP pH-Probe 590-9.0, DPP pH-Probe590-11.0, DPP pH-Probe 590-11.0, Dragon Green, DRAQ5, DsRed, DsRed,DsRed, DsRed-Express, DsRed-Express2, DsRed-Express T1, dTomato,DY-350XL, DY-480, DY-480XL MegaStokes, DY-485, DY-485XL MegaStokes,DY-490, DY-490XL MegaStokes, DY-500, DY-500XL MegaStokes, DY-520,DY-520XL MegaStokes, DY-547, DY-549P1, DY-549P1, DY-554, DY-555, DY-557,DY-557, DY-590, DY-590, DY-615, DY-630, DY-631, DY-633, DY-635, DY-636,DY-647, DY-649P1, DY-649P1, DY-650, DY-651, DY-656, DY-673, DY-675,DY-676, DY-680, DY-681, DY-700, DY-701, DY-730, DY-731, DY-750, DY-751,DY-776, DY-782, Dye-28, Dye-33, Dye-45, Dye-304, Dye-1041, DyLight 488,DyLight 549, DyLight 594, DyLight 633, DyLight 649, DyLight 680,E2-Crimson, E2-Orange, E2-Red/Green, EBFP, ECF, ECFP, ECL Plus, eGFP,ELF 97, Emerald, Envy Green, Eosin, Eosin Y, epicocconone, EqFP611,Erythrosin-5-isothiocyanate, Ethidium bromide, ethidium homodimer-1,Ethyl Eosin, Ethyl Eosin, Ethyl Nile Blue A,Ethyl-p-Dimethylaminobenzoate, Ethyl-p-Dimethylaminobenzoate, Eu2O3nanoparticles, Eu (Soini), Eu(tta)3DEADIT, EvaGreen, EVOblue-30, EYFP,FAD, FITC, FITC, FlAsH (Adams), Flash Red EX, FlAsH-CCPGCC,FlAsH-CCXXCC, Fluo-3, Fluo-4, Fluo-5F, Fluorescein, Fluorescein 0.1NaOH, Fluorescein-Dibase, fluoro-emerald, Fluorol 5G, FluoSpheres blue,FluoSpheres crimson, FluoSpheres dark red, FluoSpheres orange,FluoSpheres red, FluoSpheres yellow-green, FM4-64 in CTC, FM4-64 in SDS,FM 1-43, FM 4-64, Fort Orange 600, Fura Red, Fura Red Ca free, fura-2,Fura-2 Ca free, Gadodiamide, Gd-Dtpa-Bma, Gadodiamide, Gd-Dtpa-Bma,GelGreen™, GelRed™, H₉-40, HcRed1, Hemo Red 720, HiLyte Fluor 488,HiLyte Fluor 555, HiLyte Fluor 647, HiLyte Fluor 680, HiLyte Fluor 750,HiLyte Plus 555, HiLyte Plus 647, HiLyte Plus 750, HmGFP, Hoechst 33258,Hoechst 33342, Hoechst-33258, Hoechst-33258, Hops Yellow 560, HPTS,HPTS, HPTS, HPTS, HPTS, indo-1, Indo-1 Ca free, Ir(Cn)2(acac),Ir(Cs)2(acac), IR-775 chloride, IR-806, Ir-OEP—CO-Cl, IRDye® 650 Alkyne,IRDye® 650 Azide, IRDye® 650 Carboxylate, IRDye® 650 DBCO, IRDye® 650Maleimide, IRDye® 650 NHS Ester, IRDye® 680LT Carboxylate, IRDye® 680LTMaleimide, IRDye® 680LT NHS Ester, IRDye® 680RD Alkyne, IRDye® 680RDAzide, IRDye® 680RD Carboxylate, IRDye® 680RD DBCO, IRDye® 680RDMaleimide, TRDye® 680RD NHS Ester, IRDye® 700 phosphoramidite, IRDye®700DX, IRDye® 700DX, IRDye® 700DX Carboxylate, IRDye® 700DX NHS Ester,IRDye® 750 Carboxylate, IRDye® 750 Maleimide, IRDye® 750 NHS Ester,IRDye® 800 phosphoramidite, IRDye® 800CW, IRDye® 800CW Alkyne, IRDye®800CW Azide, IRDye® 800CW Carboxylate, IRDye® 800CW DBCO, IRDye® 800CWMaleimide, IRDye® 800CW NHS Ester, IRDye® 800RS, IRDye® 800RSCarboxylate, IRDye® 800RS NHS Ester, IRDye® QC-1 Carboxylate, IRDye®QC-1 NHS Ester, Isochrysis galbana—Parke, JC-1, JC-1, JOJO-1, JonamacRed Evitag T2, Kaede Green, Kaede Red, kusabira orange, Lake Placid 490,LDS 751, Lissamine Rhodamine (Weiss), LOLO-1, lucifer yellow CH, LuciferYellow CH, lucifer yellow CH, Lucifer Yellow CH Dilitium salt, LumioGreen, Lumio Red, Lumogen F Orange, Lumogen Red F300, Lumogen Red F300,LysoSensor Blue DND-192, LysoSensor Green DND-153, LysoSensor GreenDND-153, LysoSensor Yellow/Blue DND-160 pH 3, LysoSensor YellowBlueDND-160, LysoTracker Blue DND-22, LysoTracker Blue DND-22, LysoTrackerGreen DND-26, LysoTracker Red DND-99, LysoTracker Yellow HCK-123, MacounRed Evitag T2, Macrolex Fluorescence Red G, Macrolex Fluorescence Yellow10GN, Macrolex Fluorescence Yellow 10GN, Magnesium Green, MagnesiumOctaethylporphyrin, Magnesium Orange, Magnesium Phthalocyanine,Magnesium Phthalocyanine, Magnesium Tetramesitylporphyrin, MagnesiumTetraphenylporphyrin, malachite green isothiocyanate, Maple Red-Orange620, Marina Blue, mBanana, mBBr, mCherry, Merocyanine 540, Methyl green,Methyl green, Methyl green, Methylene Blue, Methylene Blue, mHoneyDew,MitoTracker Deep Red 633, MitoTracker Green FM, MitoTracker OrangeCMTMRos, MitoTracker Red CMXRos, monobromobimane, Monochlorobimane,Monoraphidium, mOrange, mOrange2, mPlum, mRaspberry, mRFP, mRFP1,mRFP1.2 (Wang), mStrawberry (Shaner), mTangerine (Shaner),N,N-Bis(2,4,6-trimethylphenyl)-3,4:9,10-perylenebis(dicarboximide),NADH, Naphthalene, Naphthalene, Naphthofluorescein, Naphthofluorescein,NBD-X, NeuroTrace 500525, Nilblau perchlorate, nile blue, Nile Blue,Nile Blue (EtOH), nile red, Nile Red, Nile Red, Nile red, Nileblue A,NIR1, NIR2, NIR3, NIR4, NIR820, Octaethylporphyrin, OH butoxyaza-BODIPY, OHC12 aza-BODIPY, Orange Fluorescent Protein, Oregon Green488, Oregon Green 488 DUPE, Oregon Green 514, Oxazin1, Oxazin 750,Oxazine 1, Oxazine 170, P4-3, P-Quaterphenyl, P-Terphenyl, PA-GFP(post-activation), PA-GFP (pre-activation), Pacific Orange,Palladium(II) meso-tetraphenyl-tetrabenzoporphyrin, PdOEPK, PdTFPP,PerCP-Cy5.5, Perylene, Perylene, Perylene bisimide pH-Probe 550-5.0,Perylene bisimide pH-Probe 550-5.5, Perylene bisimide pH-Probe 550-6.5,Perylene Green pH-Probe 720-5.5, Perylene Green Tag pH-Probe 720-6.0,Perylene Orange pH-Probe 550-2.0, Perylene Orange Tag 550, Perylene RedpH-Probe 600-5.5, Perylenediimid, Perylne Green pH-Probe 740-5.5,Phenol, Phenylalanine, pHrodo, succinimidyl ester, Phthalocyanine,PicoGreen dsDNA quantitation reagent, Pinacyanol-Iodide, Piroxicam,Platinum(II) tetraphenyltetrabenzoporphyrin, Plum Purple, PO-PRO-1,PO-PRO-3, POPO-1, POPO-3, POPOP, Porphin, PPO, Proflavin,PromoFluor-350, PromoFluor-405, PromoFluor-415, PromoFluor-488,PromoFluor-488 Premium, PromoFluor-488LSS, PromoFluor-500LSS,PromoFluor-505, PromoFluor-510LSS, PromoFluor-514LSS, PromoFluor-520LSS,PromoFluor-532, PromoFluor-546, PromoFluor-555, PromoFluor-590,PromoFluor-610, PromoFluor-633, PromoFluor-647, PromoFluor-670,PromoFluor-680, PromoFluor-700, PromoFluor-750, PromoFluor-770,PromoFluor-780, PromoFluor-840, propidium iodide, Protoporphyrin IX,PTIR475/UF, PTIR545/UF, PtOEP, PtOEPK, PtTFPP, Pyrene, QD525, QD565,QD585, QD605, QD655, QD705, QD800, QD903, QD PbS 950, QDot 525, QDot545, QDot 565, Qdot 585, Qdot 605, Qdot 625, Qdot 655, Qdot 705, Qdot800, QpyMe2, QSY 7, QSY 7, QSY 9, QSY 21, QSY 35, quinine, QuinineSulfate, Quinine sulfate, R-phycoerythrin, R-phycoerythrin,ReAsH-CCPGCC, ReAsH-CCXXCC, Red Beads (Weiss), Redmond Red, Resorufin,resorufin, rhod-2, Rhodamin 700 perchlorate, rhodamine, Rhodamine 6G,Rhodamine 6G, Rhodamine 101, rhodamine 110, Rhodamine 123, rhodamine123, Rhodamine B, Rhodamine B, Rhodamine Green, Rhodamine pH-Probe585-7.0, Rhodamine pH-Probe 585-7.5, Rhodamine phalloidin, RhodamineRed-X, Rhodamine Red-X, Rhodamine Tag pH-Probe 585-7.0, Rhodol Green,Riboflavin, Rose Bengal, Sapphire, SBFI, SBFI Zero Na, Scenedesmus sp.,SensiLight PBXL-1, SensiLight PBXL-3, Seta 633-NHS, Seta-633-NHS,SeTau-380-NHS, SeTau-647-NHS, Snake-Eye Red 900, SNIR1, SNIR2, SNIR3,SNIR4, Sodium Green, Solophenyl flavine 7GFE 500, Spectrum Aqua,Spectrum Blue, Spectrum FRed, Spectrum Gold, Spectrum Green, SpectrumOrange, Spectrum Red, Squarylium dye III, Stains All, Stilben derivate,Stilbene, Styryl8 perchlorate, Sulfo-Cyanine3 carboxylic acid,Sulfo-Cyanine3 carboxylic acid, Sulfo-Cyanine3 NHS ester, Sulfo-Cyanine5carboxylic acid, Sulforhodamine 101, sulforhodamine 101, SulforhodamineB, Sulforhodamine G, Suncoast Yellow, SuperGlo BFP, SuperGlo GFP, SurfGreen EX, SYBR Gold nucleic acid gel stain, SYBR Green I, SYPRO Ruby,SYTO 9, SYTO 11, SYTO 13, SYTO 16, SYTO 17, SYTO 45, SYTO 59, SYTO 60,SYTO 61, SYTO 62, SYTO 82, SYTO RNASelect, SYTO RNASelect, SYTOX Blue,SYTOX Green, SYTOX Orange, SYTOX Red, T-Sapphire, Tb (Soini), tCO,tdTomato, Terrylen, Terrylendiimid, testdye, Tetra-t-Butylazaporphine,Tetra-t-Butylnaphthalocyanine, Tetracen,Tetrakis(o-Aminophenyl)Porphyrin, Tetramesitylporphyrin,Tetramethylrhodamine, tetramethylrhodamine, Tetraphenylporphyrin,Tetraphenylporphyrin, Texas Red, Texas Red DUPE, Texas Red-X,ThiolTracker Violet, Thionin acetate, TMRE, TO-PRO-1, TO-PRO-3, Toluene,Topaz (Tsienl998), TOTO-1, TOTO-3, Tris(2,2-Bipyridyl)Ruthenium(II)chloride, Tris(4,4-diphenyl-2,2-bipyridine) ruthenium(II) chloride,Tris(4,7-diphenyl-1,10-phenanthroline) ruthenium(II) TMS, TRITC (Weiss),TRITC Dextran (Weiss), Tryptophan, Tyrosine, Vex1, Vybrant DyeCycleGreen stain, Vybrant DyeCycle Orange stain, Vybrant DyeCycle Violetstain, WEGFP (post-activation), WellRED D2, WellRED D3, WellRED D4,WtGFP, WtGFP (Tsien1998), X-rhod-1, Yakima Yellow, YFP, YO-PRO-1,YO-PRO-3, YOYO-1, YoYo-1, YoYo-1 dsDNA, YoYo-1 ssDNA, YOYO-3, ZincOctaethylporphyrin, Zinc Phthalocyanine, Zinc Tetramesitylporphyrin,Zinc Tetraphenylporphyrin, ZsGreen1, or ZsYellow1.

In embodiments, the detectable label is a fluorescent dye. Inembodiments, the detectable label is a fluorescent dye capable ofexchanging energy with another fluorescent dye (e.g., fluorescenceresonance energy transfer (FRET) chromophores).

In embodiments, the detectable moiety is a moiety of a derivative of oneof the detectable moieties described immediately above, wherein thederivative differs from one of the detectable moieties immediately aboveby a modification resulting from the conjugation of the detectablemoiety to a compound described herein.

The term “cyanine” or “cyanine moiety” as described herein refers to adetectable moiety containing two nitrogen groups separated by apolymethine chain. In embodiments, the cyanine moiety has 3 methinestructures (i.e. cyanine 3 or Cy3). In embodiments, the cyanine moietyhas 5 methine structures (i.e. cyanine 5 or Cy5). In embodiments, thecyanine moiety has 7 methine structures (i.e. cyanine 7 or Cy7).

Descriptions of nucleotide analogues of the present disclosure arelimited by principles of chemical bonding known to those skilled in theart. Accordingly, where a group may be substituted by one or more of anumber of substituents, such substitutions are selected so as to complywith principles of chemical bonding and to give compounds which are notinherently unstable and/or would be known to one of ordinary skill inthe art as likely to be unstable under ambient conditions, such asaqueous, neutral, and several known physiological conditions. Forexample, a heterocycloalkyl or heteroaryl is attached to the remainderof the molecule via a ring heteroatom in compliance with principles ofchemical bonding known to those skilled in the art thereby avoidinginherently unstable compounds.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are near each other, and, inthe case of a secretory leader, contiguous and in reading phase.However, enhancers do not have to be contiguous. Linking is accomplishedby ligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an α carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid. The terms“non-naturally occurring amino acid” and “unnatural amino acid” refer toamino acid analogs, synthetic amino acids, and amino acid mimetics,which are not found in nature.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues,wherein the polymer may In embodiments be conjugated to a moiety thatdoes not consist of amino acids. The terms apply to amino acid polymersin which one or more amino acid residue is an artificial chemicalmimetic of a corresponding naturally occurring amino acid, as well as tonaturally occurring amino acid polymers and non-naturally occurringamino acid polymers. A “fusion protein” refers to a chimeric proteinencoding two or more separate protein sequences that are recombinantlyexpressed as a single moiety.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, “conservatively modified variants” refers to those nucleicacids that encode identical or essentially identical amino acidsequences. Because of the degeneracy of the genetic code, a number ofnucleic acid sequences will encode any given protein. For instance, thecodons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, atevery position where an alanine is specified by a codon, the codon canbe altered to any of the corresponding codons described without alteringthe encoded polypeptide. Such nucleic acid variations are “silentvariations,” which are one species of conservatively modifiedvariations. Every nucleic acid sequence herein, which encodes apolypeptide, also describes every possible silent variation of thenucleic acid. One of skill will recognize that each codon in a nucleicacid (except AUG, which is ordinarily the only codon for methionine, andTGG, which is ordinarily the only codon for tryptophan) can be modifiedto yield a functionally identical molecule. Accordingly, each silentvariation of a nucleic acid that encodes a polypeptide is implicit ineach described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the disclosure.

The following groups each contain amino acids that are conservativesubstitutions for one another: 1) Non-polar—Alanine (A), Leucine (L),Isoleucine (I), Valine (V), Glycine (G), Methionine (M); 2)Aliphatic—Alanine (A), Leucine (L), Isoleucine (I), Valine (V); 3)Acidic—Aspartic acid (D), Glutamic acid (E); 4) Polar—Asparagine (N),Glutamine (Q); Serine (S), Threonine (T); 5) Basic—Arginine (R), Lysine(K); 7) Aromatic—Phenylalanine (F), Tyrosine (Y), Tryptophan (W),Histidine (H); 8) Other—Cystein (C) and Proline (P).

The term “amino acid side chain” refers to the functional substituentcontained on amino acids. For example, an amino acid side chain may bethe side chain of a naturally occurring amino acid. Naturally occurringamino acids are those encoded by the genetic code (e.g., alanine,arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid,glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine, orvaline), as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. In embodiments,the amino acid side chain may be a non-natural amino acid side chain. Inembodiments, the amino acid side chain is H,

The term “non-natural amino acid side chain” refers to the functionalsubstituent of compounds that have the same basic chemical structure asa naturally occurring amino acid, i.e., an α carbon that is bound to ahydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium, allylalanine, 2-aminoisobutryric acid. Non-natural aminoacids are non-proteinogenic amino acids that occur naturally or arechemically synthesized. Such analogs have modified R groups (e.g.,norleucine) or modified peptide backbones, but retain the same basicchemical structure as a naturally occurring amino acid. Non-limitingexamples include exo-cis-3-Aminobicyclo[2.2.1]hept-5-ene-2-carboxylicacid hydrochloride, cis-2-Aminocycloheptanecarboxylic acidhydrochloride,cis-6-Amino-3-cyclohexene-1-carboxylic acid hydrochloride,cis-2-Amino-2-methylcyclohexanecarboxylic acid hydrochloride,cis-2-Amino-2-methylcyclopentanecarboxylic acid hydrochloride,2-(Boc-aminomethyl)benzoic acid, 2-(Boc-amino)octanedioic acid,Boc-4,5-dehydro-Leu-OH (dicyclohexylammonium),Boc-4-(Fmoc-amino)-L-phenylalanine, Boc-β-Homopyr-OH,Boc-(2-indanyl)-Gly-OH, 4-Boc-3-morpholineacetic acid,4-Boc-3-morpholineacetic acid, Boc-pentafluoro-D-phenylalanine,Boc-pentafluoro-L-phenylalanine, Boc-Phe(2-Br)—OH, Boc-Phe(4-Br)—OH,Boc-D-Phe(4-Br)—OH, Boc-D-Phe(3-Cl)—OH, Boc-Phe(4-NH2)-OH,Boc-Phe(3-NO2)-OH, Boc-Phe(3,5-F2)-OH,2-(4-Boc-piperazino)-2-(3,4-dimethoxyphenyl)acetic acid purum,2-(4-Boc-piperazino)-2-(2-fluorophenyl)acetic acid purum,2-(4-Boc-piperazino)-2-(3-fluorophenyl)acetic acid purum,2-(4-Boc-piperazino)-2-(4-fluorophenyl)acetic acid purum,2-(4-Boc-piperazino)-2-(4-methoxyphenyl)acetic acid purum,2-(4-Boc-piperazino)-2-phenylacetic acid purum,2-(4-Boc-piperazino)-2-(3-pyridyl)acetic acid purum,2-(4-Boc-piperazino)-2-[4-(trifluoromethyl)phenyl]acetic acid purum,Boc-β-(2-quinolyl)-Ala-OH, N-Boc-1,2,3,6-tetrahydro-2-pyridinecarboxylicacid, Boc-β-(4-thiazolyl)-Ala-OH, Boc-β-(2-thienyl)-D-Ala-OH,Fmoc-N-(4-Boc-aminobutyl)-Gly-OH, Fmoc-N-(2-Boc-aminoethyl)-Gly-OH,Fmoc-N-(2,4-dimethoxybenzyl)-Gly-OH, Fmoc-(2-indanyl)-Gly-OH,Fmoc-pentafluoro-L-phenylalanine, Fmoc-Pen(Trt)-OH, Fmoc-Phe(2-Br)—OH,Fmoc-Phe(4-Br)—OH, Fmoc-Phe(3,5-F2)-OH, Fmoc-β-(4-thiazolyl)-Ala-OH,Fmoc-β-(2-thienyl)-Ala-OH, 4-(Hydroxymethyl)-D-phenylalanine.

“Percentage of sequence identity” is determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide or polypeptide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) as compared tothe reference sequence for optimal alignment of the two sequences. Thepercentage is calculated by determining the number of positions at whichthe identical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over aspecified region, when compared and aligned for maximum correspondenceover a comparison window or designated region) as measured using a BLASTor BLAST 2.0 sequence comparison algorithms with default parametersdescribed below, or by manual alignment and visual inspection (see,e.g., NCBI web site www.ncbi.nlm.nih.gov/BLAST/ or the like). Suchsequences are then said to be “substantially identical.” This definitionalso refers to, or may be applied to, the compliment of a test sequence.The definition also includes sequences that have deletions and/oradditions, as well as those that have substitutions. As described below,the preferred algorithms can account for gaps and the like. Preferably,identity exists over a region that is at least about 25 amino acids ornucleotides in length, or more preferably over a region that is 50-100amino acids or nucleotides in length. As used herein, percent (%) aminoacid sequence identity is defined as the percentage of amino acids in acandidate sequence that are identical to the amino acids in a referencesequence, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity. Alignmentfor purposes of determining percent sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN,ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters formeasuring alignment, including any algorithms needed to achieve maximalalignment over the full-length of the sequences being compared can bedetermined by known methods.

For sequence comparisons, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Preferably,default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 10 to 700, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., CurrentProtocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

An amino acid or nucleotide base “position” is denoted by a number thatsequentially identifies each amino acid (or nucleotide base) in thereference sequence based on its position relative to the N-terminus (or5′-end). Due to deletions, insertions, truncations, fusions, and thelike that must be taken into account when determining an optimalalignment, in general the amino acid residue number in a test sequencedetermined by simply counting from the N-terminus will not necessarilybe the same as the number of its corresponding position in the referencesequence. For example, in a case where a variant has a deletion relativeto an aligned reference sequence, there will be no amino acid in thevariant that corresponds to a position in the reference sequence at thesite of deletion. Where there is an insertion in an aligned referencesequence, that insertion will not correspond to a numbered amino acidposition in the reference sequence. In the case of truncations orfusions there can be stretches of amino acids in either the reference oraligned sequence that do not correspond to any amino acid in thecorresponding sequence.

The term “DNA polymerase” and “nucleic acid polymerase” are used inaccordance with their plain ordinary meaning and refer to enzymescapable of synthesizing nucleic acid molecules from nucleotides (e.g.,deoxyribonucleotides). Typically, a DNA polymerase adds nucleotides tothe 3′-end of a DNA strand, one nucleotide at a time. In embodiments,the DNA polymerase is a Pol I DNA polymerase, Pol II DNA polymerase, PolIII DNA polymerase, Pol IV DNA polymerase, Pol V DNA polymerase, Pol βDNA polymerase, Pol μ DNA polymerase, Pol λ DNA polymerase, Pol σ DNApolymerase, Pol α DNA polymerase, Pol δ DNA polymerase, Pol ε DNApolymerase, Pol η DNA polymerase, Pol ι DNA polymerase, Pol κ DNApolymerase, Pol ζ DNA polymerase, Pol γ DNA polymerase, Pol θ DNApolymerase, Pol υ DNA polymerase, or a thermophilic nucleic acidpolymerase (e.g. Therminator γ, 9° N polymerase (exo-), Therminator II,Therminator III, or Therminator IX). In embodiments, the DNA polymeraseis a Pyrococcus DNA polymerase.

The term “thermophilic nucleic acid polymerase” as used herein refers toa family of DNA polymerases (e.g., 9° N™) and mutants thereof derivedfrom the DNA polymerase originally isolated from the hyperthermophilicarchaea, Thermococcus sp. 9 degrees N-7, found in hydrothermal vents atthat latitude (East Pacific Rise) (Southworth M W, et al. PNAS. 1996;93(11):5281-5285). A thermophilic nucleic acid polymerase is a member ofthe family B DNA polymerases. Site-directed mutagenesis of the 3′-5′ exomotif I (Asp-Ile-Glu or DIE) to AIA, AIE, EIE, EID or DIA yieldedpolymerase with no detectable 3′ exonuclease activity. Mutation toAsp-Ile-Asp (DID) resulted in reduction of 3′-5′ exonuclease specificactivity to <1% of wild type, while maintaining other properties of thepolymerase including its high strand displacement activity. The sequenceAIA (D141A, E143A) was chosen for reducing exonuclease. Subsequentmutagenesis of key amino acids results in an increased ability of theenzyme to incorporate dideoxynucleotides, ribonucleotides andacyclonucleotides (e.g., Therminator II enzyme from New England Biolabswith D141A/E143A/Y409V/A485L mutations); 3′-amino-dNTPs, 3′-azido-dNTPsand other 3′-modified nucleotides (e.g., NEB Therminator III DNAPolymerase with D141A/E143A/L408S/Y409A/P410V mutations, NEB TherminatorIX DNA polymerase), or γ-phosphate labeled nucleotides (e.g.,Therminator γ:D141A/E143A/W355A/L408W/R460A/Q461S/K464E/D480V/R484W/A485L). Typically,these enzymes do not have 5′-3′ exonuclease activity. Additionalinformation about thermophilic nucleic acid polymerases may be found in(Southworth M W, et al. PNAS. 1996; 93(11):5281-5285; Bergen K, et al.ChemBioChem. 2013; 14(9):1058-1062; Kumar S, et al. Scientific Reports.2012; 2:684; Fuller C W, et al. 2016; 113(19):5233-5238; Guo J, et al.Proceedings of the National Academy of Sciences of the United States ofAmerica. 2008; 105(27):9145-9150), which are incorporated herein intheir entirety for all purposes.

In the context of this application, the term “motif A region”specifically refers to the three amino acids functionally equivalent orhomologous to amino acids 409, 410, and 411 in wild type P. horikoshii;these amino acids are functionally equivalent to amino acid positions408, 409, and 410 in 9° N polymerase. Functionally equivalent orhomologous “motif A regions” of polymerases other than P. horikoshii canbe identified on the basis of amino acid sequence alignment and/ormolecular modeling. Sequence alignments may be compiled using any of thestandard alignment tools known in the art, such as for example BLASTetc.

The term “exonuclease activity” is used in accordance with its ordinarymeaning in the art, and refers to the removal of a nucleotide from anucleic acid by a DNA polymerase. For example, during polymerization,nucleotides are added to the 3′ end of the primer strand. Occasionally aDNA polymerase incorporates an incorrect nucleotide to the 3′-OHterminus of the primer strand, wherein the incorrect nucleotide cannotform a hydrogen bond to the corresponding base in the template strand.Such a nucleotide, added in error, is removed from the primer as aresult of the 3′ to 5′ exonuclease activity of the DNA polymerase. Inembodiments, exonuclease activity may be referred to as “proofreading.”When referring to 3′-5′ exonuclease activity, it is understood that theDNA polymerase facilitates a hydrolyzing reaction that breaksphosphodiester bonds at either the 3′ end of a polynucleotide chain toexcise the nucleotide. In embodiments, 3′-5′ exonuclease activity refersto the successive removal of nucleotides in single-stranded DNA in a3′→5′ direction, releasing deoxyribonucleoside 5′-monophosphates oneafter another. Methods for quantifying exonuclease activity are known inthe art, see for example Southworth et al, PNAS Vol 93, 8281-8285(1996).

The term “independently resolvable” means that the entity under studycan be examined independent of other entities due to spatial separation,for example, one polymerase-template complex can be probed independentfrom another polymerase-template complex present at a different physicallocation, or “address”, on the solid substrate.

The terms “measure”, “measuring”, “measurement” and the like refer notonly to quantitative measurement of a particular variable, but also toqualitative and semi-quantitative measurements. Accordingly,“measurement” also includes detection, meaning that merely detecting achange, without quantification, constitutes measurement.

The term “population” refers to a collection of one or more entities,e.g., DNA molecules.

“Perfectly matched” in reference to a nucleic acid duplex means that thepoly- or oligonucleotide strands making up the duplex form adouble-stranded structure, or region of double-stranded structure, withone another such that every nucleotide (or nucleotide analogue) in eachstrand undergoes Watson-Crick base-pairing with a nucleotide in theother strand in the duplexed (i.e., hybridized) region. The term alsocomprehends the pairing of nucleoside analogues, such as deoxyinosinewith deoxycytidine, and the like. Conversely, a “mismatch” in a nucleicacid duplex means that one or more pairs of nucleotides in the duplexfail to undergo Watson-Crick base-pairing.

A “polymerase-template complex” refers to functional complex between aDNA polymerase and a DNA primer-template molecule (e.g., nucleic acid)being sequenced.

The terms “sequencing”, “sequence determination”, “determining anucleotide sequence”, and the like include determination of partial aswell as full sequence information of the polynucleotide being sequenced.That is, the term includes sequence comparisons, fingerprinting, andlike levels of information about a target polynucleotide, as well as theexpress identification and ordering of nucleotides in a targetpolynucleotide. The term also includes the determination of theidentification, ordering, and locations of one, two, or three of thefour types of nucleotides within a target polynucleotide.

The term “sequencing reaction mixture” refers to an aqueous mixture thatcontains the reagents necessary to allow a dNTP or dNTP analogue to adda nucleotide to a DNA strand by a DNA polymerase. Exemplary mixturesinclude buffers (e.g., saline-sodium citrate (SSC),tris(hydroxymethyl)aminomethane or “Tris”), salts (e.g., KCl or(NH₄)₂SO₄)), nucleotides, polymerases, cleaving agent (e.g.,tri-n-butyl-phosphine, triphenyl phosphine and its sulfonated versions(i.e., tris(3-sulfophenyl)-phosphine, TPPTS), andtri(carboxyethyl)phosphine (TCEP) and its salts, cleaving agentscavenger compounds (e.g., 2′-Dithiobisethanamine or11-Azido-3,6,9-trioxaundecane-1-amine), detergents and/or crowdingagents or stabilizers (e.g., PEG, Tween, BSA).

The term “solid substrate” means any suitable medium present in thesolid phase to which an antibody or an agent can be covalently ornon-covalently affixed or immobilized. Preferred solid substrates areglass. Non-limiting examples include chips, beads and columns. The solidsubstrate can be non-porous or porous. Exemplary solid substratesinclude, but are not limited to, glass and modified or functionalizedglass, plastics (including acrylics, polystyrene and copolymers ofstyrene and other materials, polypropylene, polyethylene, polybutylene,polyurethanes, Teflon™, cyclic olefins, polyimides, etc.), nylon,ceramics, resins, Zeonor, silica or silica-based materials includingsilicon and modified silicon, carbon, metals, inorganic glasses, opticalfiber bundles, and polymers.

The term “species”, when used in the context of describing a particularcompound or molecule species, refers to a population of chemicallyindistinct molecules. When used in the context of taxonomy, “species” isthe basic unit of classification and a taxonomic rank. For example, inreference to the microorganism Pyrococcus horikoshii, horikoshii is aspecies of the genus Pyrococcus.

The terms “position”, “numbered with reference to” or “correspondingto,” when used in the context of the numbering of a given amino acid orpolynucleotide sequence, refer to the numbering of the residues of aspecified reference sequence when the given amino acid or polynucleotidesequence is compared to the reference sequence. As used herein, the term“functionally equivalent to” in relation to an amino acid positionrefers to an amino acid residue in a protein that corresponds to aparticular amino acid in a reference sequence. An amino acid“corresponds” to a given residue when it occupies the same essentialstructural position within the protein as the given residue. One skilledin the art will immediately recognize the identity and location ofresidues corresponding to a specific position in a protein (e.g.,polymerase) in other proteins with different numbering systems. Forexample, by performing a simple sequence alignment with a protein (e.g.,polymerase) the identity and location of residues corresponding tospecific positions of said protein are identified in other proteinsequences aligning to said protein. For example, a selected residue in aselected protein corresponds to methionine at position 129 when theselected residue occupies the same essential spatial or other structuralrelationship as a methionine at position 129. In some embodiments, wherea selected protein is aligned for maximum homology with a protein, theposition in the aligned selected protein aligning with methionine 129said to correspond to methionine 129. Instead of a primary sequencealignment, a three dimensional structural alignment can also be used,e.g., where the structure of the selected protein is aligned for maximumcorrespondence with the methionine at position 129, and the overallstructures compared. In this case, an amino acid that occupies the sameessential position as methionine 129 in the structural model is said tocorrespond to the methionine 129 residue. For example, references to aP. horikoshii polymerase amino acid position recited herein may refer toa numbered position set forth in SEQ ID NO:1, or the correspondingposition in a polymerase homolog of SEQ ID NO:1. In embodiments,references to a polymerase amino acid position recited herein refers toa numbered position set forth in SEQ ID NO:1 which is the amino acidsequence of the wild type P. horikoshii polymerase. In embodiments, thepolymerase provided herein may have one or more amino acid substitutionmutations at position 129, 141, 142, 143, 144, 153, 215, 315, 477, 478,479, 486, 515, 522, 591, 603, 640, 713, 714, 719, 720, and/or 736 wherethe numbering is in reference to the amino acid position as provided inSEQ ID NO: 1. In embodiments, the polymerase provided herein may includemutations at positions in a parent polymerase corresponding to positionsin SEQ ID NO: 1 identified as follows: 141, 143, 409, 410, and 411. Inembodiments, the polymerase provided herein may include mutations atpositions in a parent polymerase corresponding to positions in SEQ IDNO: 1 identified as follows: 141, 143, 409, 410, and 411 and furtherinclude one or more mutations at positions 144, 153, 215, 315, 477, 478,479, 486, 515, 522, 591, 603, 640, 713, 714, 719, 720, and 736. Inembodiments, the polymerase provided herein may include mutations atpositions in a parent polymerase corresponding to positions in SEQ IDNO: 1 identified as follows: 129, 141, 143, 409, 410, and 411. Inembodiments, the polymerase provided herein may include mutations atpositions in a parent polymerase corresponding to positions in SEQ IDNO: 1 identified as follows: 129, 141, 143, 409, 410, and 411 andfurther include one or more mutations at positions 144, 153, 215, 315,477, 478, 479, 515, 522, 591, 603, 640, 713, 714, 719, 720, and 736. Inembodiments, the polymerase provided herein may include mutations atpositions in a parent polymerase corresponding to positions in SEQ IDNO: 1 identified as follows: 141, 143, 153, 409, 410, and 411. Inembodiments, the polymerase provided herein may include mutations atpositions in a parent polymerase corresponding to positions in SEQ IDNO: 1 identified as follows: 141, 143, 153, 409, 410, and 411 andfurther include one or more mutations at positions 144, 215, 315, 477,478, 479, 515, 522, 591, 603, 640, 713, 714, 719, 720, and 736. Inembodiments, the polymerase provided herein may include mutations atpositions in a parent polymerase corresponding to positions in SEQ IDNO: 1 identified as follows: 129, 141, 143, 153, 409, 410, 411, and 486.In embodiments, the polymerase provided herein may include mutations atpositions in a parent polymerase corresponding to positions in SEQ IDNO: 1 identified as follows: 129, 141, 143, 153, 409, 410, 411, and 486and further include one or more mutations at positions 144, 215, 315,477, 478, 479, 515, 522, 591, 603, 640, 713, 714, 719, 720, and 736.

In embodiments, the polymerase may include an amino acid substitutionmutation at a particular position corresponding to a position in SEQ IDNO: 1. For example, in embodiments, the polymerase includes an aminoacid substitution mutation at position 141, which means the variantpolymerase has a different amino acid at position 141 compared to SEQ IDNO: 1. In embodiments, the polymerase includes an amino acidsubstitution mutation at more than one position compared to SEQ IDNO: 1. For example, in embodiments, the polymerase includes thefollowing substitution mutations: D141A; E143A; L409S; Y410A; P411V,where the number refers to the corresponding position in SEQ ID NO: 1.One having skill in the art would understand the amino acid mutationnomenclature, such that D141A refers to aspartic acid (single lettercode is D), at position 141, is replaced with alanine (single lettercode A).

The terms “agonist,” “activator,” “upregulator,” etc. refer to asubstance capable of detectably increasing the expression or activity ofa given gene or protein. The agonist can increase expression or activity10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to acontrol in the absence of the agonist. In certain instances, expressionor activity is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold orhigher than the expression or activity in the absence of the agonist.

As defined herein, the term “inhibition”, “inhibit”, “inhibiting” andthe like in reference to a protein-inhibitor interaction meansnegatively affecting (e.g. decreasing) the activity or function of theprotein relative to the activity or function of the protein in theabsence of the inhibitor. In embodiments inhibition means negativelyaffecting (e.g. decreasing) the concentration or levels of the proteinrelative to the concentration or level of the protein in the absence ofthe inhibitor. In embodiments, inhibition refers to reduction of adisease or symptoms of disease. In embodiments, inhibition refers to areduction in the activity of a particular protein target. Thus,inhibition includes, at least in part, partially or totally blockingstimulation, decreasing, preventing, or delaying activation, orinactivating, desensitizing, or down-regulating signal transduction orenzymatic activity or the amount of a protein. In embodiments,inhibition refers to a reduction of activity of a target proteinresulting from a direct interaction (e.g. an inhibitor binds to thetarget protein). In embodiments, inhibition refers to a reduction ofactivity of a target protein from an indirect interaction (e.g. aninhibitor binds to a protein that activates the target protein, therebypreventing target protein activation).

The terms “inhibitor,” “repressor” or “antagonist” or “downregulator”interchangeably refer to a substance capable of detectably decreasingthe expression or activity of a given gene or protein. The antagonistcan decrease expression or activity 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90% or more in comparison to a control in the absence of theantagonist. In certain instances, expression or activity is 1.5-fold,2-fold, 3-fold, 4-fold, 5-fold, 10-fold or lower than the expression oractivity in the absence of the antagonist.

The term “expression” includes any step involved in the production ofthe polypeptide including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion. Expression can be detected usingconventional techniques for detecting protein (e.g., ELISA, Westernblotting, flow cytometry, immunofluorescence, immunohistochemistry,etc.).

An “effective amount” is an amount sufficient for a compound toaccomplish a stated purpose relative to the absence of the compound(e.g. achieve the effect for which it is administered, treat a disease,reduce enzyme activity, increase enzyme activity, reduce a signalingpathway, or reduce one or more symptoms of a disease or condition). An“activity decreasing amount,” as used herein, refers to an amount ofantagonist required to decrease the activity of an enzyme relative tothe absence of the antagonist. A “function disrupting amount,” as usedherein, refers to the amount of antagonist required to disrupt thefunction of an enzyme or protein relative to the absence of theantagonist.

A “cell” as used herein, refers to a cell carrying out metabolic orother function sufficient to preserve or replicate its genomic DNA. Acell can be identified by well-known methods in the art including, forexample, presence of an intact membrane, staining by a particular dye,ability to produce progeny or, in the case of a gamete, ability tocombine with a second gamete to produce a viable offspring. Cells mayinclude prokaryotic and eukaryotic cells. Prokaryotic cells include butare not limited to bacteria. Eukaryotic cells include but are notlimited to yeast cells and cells derived from plants and animals, forexample mammalian, insect (e.g., spodoptera) and human cells.

“Control” or “control experiment” is used in accordance with its plainordinary meaning and refers to an experiment in which the subjects(e.g., enzymes) or reagents of the experiment are treated as in aparallel experiment except for omission of a procedure, reagent, orvariable of the experiment (e.g., a polymerase not having one or moremutations relative to the polymerase being tested). In some instances,the control is used as a standard of comparison in evaluatingexperimental effects. In some embodiments, a control is the measurementof the activity of a protein in the absence of a mutation as describedherein (including embodiments and examples). “Control polymerase” isdefined herein as the polymerase against which the activity of thealtered polymerase is compared. In one embodiment of the invention thecontrol polymerase may comprise a wild type polymerase or an exo-variantthereof. Unless otherwise stated, by “wild type” it is generally meantthat the polymerase comprises its natural amino acid sequence, as itwould be found in nature. The invention is not limited to merely acomparison of activity of the polymerases as described herein againstthe wild type equivalent or exo-variant of the polymerase that is beingaltered. Many polymerases exist whose amino acid sequence have beenmodified (e.g., by amino acid substitution mutations) and which canprove to be a suitable control for use in assessing the modifiednucleotide incorporation efficiencies of the polymerases as describedherein. The control polymerase can, therefore, comprise any knownpolymerase, including mutant polymerases known in the art. The activityof the chosen “control” polymerase with respect to incorporation of thedesired nucleotide analogues may be determined by an incorporationassay.

The term “modulate” is used in accordance with its plain ordinarymeaning and refers to the act of changing or varying one or moreproperties. “Modulation” refers to the process of changing or varyingone or more properties.

The term “kit” is used in accordance with its plain ordinary meaning andrefers to any delivery system for delivering materials or reagents forcarrying out a method of the invention. Such delivery systems includesystems that allow for the storage, transport, or delivery of reactionreagents (e.g., nucleotides, enzymes, nucleic acid templates, etc. inthe appropriate containers) and/or supporting materials (e.g., buffers,written instructions for performing the reaction, etc.) from onelocation to another location. For example, kits include one or moreenclosures (e.g., boxes) containing the relevant reaction reagentsand/or supporting materials. Such contents may be delivered to theintended recipient together or separately. For example, a firstcontainer may contain an enzyme, while a second container containsnucleotides. In embodiments, the kit includes vessels containing one ormore enzymes, primers, adaptors, or other reagents as described herein.Vessels may include any structure capable of supporting or containing aliquid or solid material and may include, tubes, vials, jars,containers, tips, etc. In embodiments, a wall of a vessel may permit thetransmission of light through the wall. In embodiments, the vessel maybe optically clear. The kit may include the enzyme and/or nucleotides ina buffer. In embodiments, the buffer includes an acetate buffer,3-(N-morpholino)propanesulfonic acid (MOPS) buffer,N-(2-Acetamido)-2-aminoethanesulfonic acid (ACES) buffer,phosphate-buffered saline (PBS) buffer,4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer,N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid(AMPSO) buffer, borate buffer (e.g., borate buffered saline, sodiumborate buffer, boric acid buffer), 2-Amino-2-methyl-1,3-propanediol(AMPD) buffer, N-cyclohexyl-2-hydroxyl-3-aminopropanesulfonic acid(CAPSO) buffer, 2-Amino-2-methyl-1-propanol (AMP) buffer,4-(Cyclohexylamino)-1-butanesulfonic acid (CABS) buffer, glycine-NaOHbuffer, N-Cyclohexyl-2-aminoethanesulfonic acid (CHES) buffer,tris(hydroxymethyl)aminomethane (Tris) buffer, or aN-cyclohexyl-3-aminopropanesulfonic acid (CAPS) buffer. In embodiments,the buffer is a borate buffer. In embodiments, the buffer is a CHESbuffer.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly indicates otherwise, between the upper and lowerlimit of that range, and any other stated or unstated intervening valuein, or smaller range of values within, that stated range is encompassedwithin the invention. The upper and lower limits of any such smallerrange (within a more broadly recited range) may independently beincluded in the smaller ranges, or as particular values themselves, andare also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

A “patentable” process, machine, or article of manufacture according tothe invention means that the subject matter satisfies all statutoryrequirements for patentability at the time the analysis is performed.For example, with regard to novelty, non-obviousness, or the like, iflater investigation reveals that one or more claims encompass one ormore embodiments that would negate novelty, non-obviousness, etc., theclaim(s), being limited by definition to “patentable” embodiments,specifically exclude the unpatentable embodiment(s). In addition, theclaims appended hereto are to be interpreted both to provide thebroadest reasonable scope, as well as to preserve their validity.Furthermore, if one or more of the statutory requirements forpatentability are amended or if the standards change for assessingwhether a particular statutory requirement for patentability issatisfied from the time this application is filed or issues as a patentto a time the validity of one or more of the appended claims isquestioned, the claims are to be interpreted in a way that (1) preservestheir validity and (2) provides the broadest reasonable interpretationunder the circumstances.

The phrase “stringent hybridization conditions” refers to conditionsunder which a primer will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acids, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength pH. The T_(m) is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Stringent conditions mayalso be achieved with the addition of destabilizing agents such asformamide. For selective or specific hybridization, a positive signal isat least two times background, preferably 10 times backgroundhybridization. Exemplary stringent hybridization conditions can be asfollowing: 50% formamide, 5×SSC, and 1% SDS, incubating at 42° C., or,5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDSat 65° C.

II. Polymerases

Provided herein are, inter alia, modified Pyrococcus Family B DNApolymerases. Family B polymerases characteristically have separatedomains for DNA polymerase activity and 3′-5′ exonuclease activity. Theexonuclease domain is characterized by as many as six and at least threeconserved amino acid sequence motifs in and around a structural bindingpocket. During polymerization, nucleotides are added to the 3′ end ofthe primer strand and during the 3′-5′ exonuclease reaction, the 3′terminus of the primer is shifted to the 3′-5′ exonuclease domain andthe one or more of the 3′-terminal nucleotides are hydrolyzed.

In embodiments, the variants of a Pyrococcus family B DNA polymeraseprovided herein have no detectable exonuclease activity and are usefulin methods of incorporating modified nucleotides in nucleic acidsynthesis reactions. In embodiments, the polymerase is a thermophilicnucleic acid polymerase.

In embodiments, the variants of a Pyrococcus family B DNA polymerase arederived from a Pyrococcus species. In embodiments, the Pyrococcusspecies include Pyrococcus abyssi, Pyrococcus endeavori, Pyrococcusfuriosus, Pyrococcus glycovorans, Pyrococcus horikoshii, Pyrococcuskukulkanii, Pyrococcus woesei, Pyrococcus yayanosii, Pyrococcus sp.,Pyrococcus sp. 1211, Pyrococcus sp. 121, Pyrococcus sp. 303, Pyrococcussp. 304, Pyrococcus sp. 312, Pyrococcus sp. 32-4, Pyrococcus sp. 321,Pyrococcus sp. 322, Pyrococcus sp. 323, Pyrococcus sp. 324, Pyrococcussp. 95-12-1, Pyrococcus sp. AV5, Pyrococcus sp. Ax99-7, Pyrococcus sp.C2, Pyrococcus sp. EX2, Pyrococcus sp. Fla95-Pc, Pyrococcus sp. GB-3A,Pyrococcus sp. GB-D, Pyrococcus sp. GBD, Pyrococcus sp. GI-H, Pyrococcussp. GI-J, Pyrococcus sp. GIL, Pyrococcus sp. HT3, Pyrococcus sp. JT1,Pyrococcus sp. LMO-A29, Pyrococcus sp. LMO-A30, Pyrococcus sp. LMO-A31,Pyrococcus sp. LMO-A32, Pyrococcus sp. LMO-A33, Pyrococcus sp. LMO-A34,Pyrococcus sp. LMO-A35, Pyrococcus sp. LMO-A36, Pyrococcus sp. LMO-A37,Pyrococcus sp. LMO-A38, Pyrococcus sp. LMO-A39, Pyrococcus sp. LMO-A40,Pyrococcus sp. LMO-A41, Pyrococcus sp. LMO-A42, Pyrococcus sp. M24D13,Pyrococcus sp. MA2.31, Pyrococcus sp. MA2.32, Pyrococcus sp. MA2.34,Pyrococcus sp. MV1019, Pyrococcus sp. MV4, Pyrococcus sp. MV7,Pyrococcus sp. MZ14, Pyrococcus sp. MZ4, Pyrococcus sp. NA2, Pyrococcussp. NS102-T, Pyrococcus sp. P12.1, Pyrococcus sp. Pikanate 5017,Pyrococcus sp. PK 5017, Pyrococcus sp. ST04, Pyrococcus sp. ST700,Pyrococcus sp. Tc-2-70, Pyrococcus sp. Tc95-7C-I, Pyrococcus sp.TC95-7C-S, Pyrococcus sp. Tc95_6, Pyrococcus sp. V211, Pyrococcus sp.V212, Pyrococcus sp. V221, Pyrococcus sp. V222, Pyrococcus sp. V231,Pyrococcus sp. V232, Pyrococcus sp. V61, Pyrococcus sp. V62, Pyrococcussp. V63, Pyrococcus sp. V72, Pyrococcus sp. V73, Pyrococcus sp. VB112,Pyrococcus sp. VB113, Pyrococcus sp. VB81, Pyrococcus sp. VB82,Pyrococcus sp. VB83, Pyrococcus sp. VB85, Pyrococcus sp. VB86,Pyrococcus sp. VB93 polymerase, Pyrococcus furiosus DSM 3638, Pyrococcussp. GE23, Pyrococcus sp. GI-H, Pyrococcus sp. NA2, Pyrococcus sp. ST04,or Pyrococcus sp. ST700.

In embodiments, the variants of a Pyrococcus family B DNA polymeraseprovided herein are a Pyrococcus horikoshii family B DNA polymerase thathas no detectable exonuclease activity and are useful in methods ofincorporating modified nucleotides in nucleic acid synthesis reactions.In embodiments, the polymerase is a thermophilic nucleic acidpolymerase.

In embodiments, the variants of a Pyrococcus family B DNA polymeraseprovided herein are a Pyrococcus abyssi family B DNA polymerase that hasno detectable exonuclease activity and are useful in methods ofincorporating modified nucleotides in nucleic acid synthesis reactions.In embodiments, the polymerase is a thermophilic nucleic acidpolymerase.

Parent archaeal polymerases may be DNA polymerases that are isolatedfrom naturally occurring organisms. The parent DNA polymerases, alsoreferred to as wild type polymerase, share the property of having astructural binding pocket that binds and hydrolyzes a substrate nucleicacid, producing 5′-dNMP. The structural binding pocket in this family ofpolymerases also shares the property of having sequence motifs that formthe binding pocket, referred to as Exo Motifs I-VI. In embodiments, theparent or wild type P. horikoshii polymerase has an amino acid sequencecomprising SEQ ID NO: 1. In embodiments, the variant P. horikoshiipolymerase has one or more amino acid substitution mutations relative toSEQ ID NO: 1. In embodiments, the parent or wild type P. abyssipolymerase has an amino acid sequence comprising SEQ ID NO: 21. Inembodiments, the variant P. abyssi polymerase has one or more amino acidsubstitution mutations relative to SEQ ID NO: 21.

“Synthetic” DNA polymerases refer to non-naturally occurring DNApolymerases such as those constructed by synthetic methods, mutatedparent DNA polymerases such as truncated DNA polymerases and fusion DNApolymerases (e.g. U.S. Pat. No. 7,541,170). Variants of the parent DNApolymerase have been engineered by mutating residues using site-directedor random mutagenesis methods known in the art. In embodiments, themutations are in any of Motifs I-VI. The variant is expressed in anexpression system such as E. coli by methods known in the art. Thevariant is then screened using the assays described herein to determineexonuclease activity.

In embodiments, the polymerase (a synthetic or variant DNA polymerase)provided herein may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, or more mutations as compared to the wild-type sequence of P.horikoshii family B DNA polymerase of SEQ ID NO: 1. The polymerase (asynthetic or variant DNA polymerase) may contain 10, 20, 30, 40, 50 ormore mutations as compared to the wild-type sequence of P. horikoshiifamily B DNA polymerase of SEQ ID NO: 1. The polymerase (a synthetic orvariant DNA polymerase) may contain between 10 and 20, between 20 and30, between 30 and 40, or between 40 or 50 mutations as compared to thewild-type sequence of P. horikoshii family B DNA polymerase of SEQ IDNO: 1.

In embodiments, the polymerase (a synthetic or variant DNA polymerase)provided herein may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, or more mutations as compared to the wild-type sequence of P.abyssi family B DNA polymerase of SEQ ID NO: 21. The polymerase (asynthetic or variant DNA polymerase) may contain 10, 20, 30, 40, 50 ormore mutations as compared to the wild-type sequence of P. abyssi familyB DNA polymerase of SEQ ID NO: 21. The polymerase (a synthetic orvariant DNA polymerase) may contain between 10 and 20, between 20 and30, between 30 and 40, or between 40 or 50 mutations as compared to thewild-type sequence of P. abyssi family B DNA polymerase of SEQ ID NO:21.

In an aspect, the polymerase (a synthetic or variant DNA polymerase)provided herein may have one or more amino acid substitution mutationsbetween positions 129 and 736 inclusive of endpoint positions. Inembodiments, the polymerase (a synthetic or variant DNA polymerase)provided herein may have one or more amino acid substitution mutationsat position 129, 141, 143, 144, 153, 215, 315, 409, 410, 411, 477, 478,479, 486, 515, 522, 591, 603, 640, 713, 714, 719, and 736. Inembodiments, the polymerase (a synthetic or variant DNA polymerase)provided herein may have one or more amino acid substitution mutationsat position 129, 141, 143, 144, 153, 215, 315, 409, 410, 411, 477, 478,479, 486, 515, 522, 591, 603, 640, 713, 714, 719, or 736.

In an aspect is provided a polymerase including an amino acid sequencethat is at least 80% identical to a continuous 500 amino acid sequencewithin SEQ ID NO: 1. In embodiments, the polymerase includes an aminoacid sequence that is at least 85% identical to a continuous 500 aminoacid sequence within SEQ ID NO: 1. In embodiments, the polymeraseincludes an amino acid sequence that is at least 90% identical to acontinuous 500 amino acid sequence within SEQ ID NO: 1. In embodiments,the polymerase includes an amino acid sequence that is at least 95%identical to a continuous 500 amino acid sequence within SEQ ID NO: 1.In embodiments, the polymerase includes an amino acid sequence that isat least 98% identical to a continuous 500 amino acid sequence withinSEQ ID NO: 1. In embodiments, the polymerase includes an amino acidsequence that is at least 99% identical to a continuous 500 amino acidsequence within SEQ ID NO: 1. In embodiments, the polymerase includes anamino acid sequence that is 90% identical to a continuous 500 amino acidsequence within SEQ ID NO: 1. In embodiments, the polymerase includes anamino acid sequence that is 95% identical to a continuous 500 amino acidsequence within SEQ ID NO: 1. In embodiments, the polymerase includes anamino acid sequence that is 90% identical to SEQ ID NO: 1. Inembodiments, the polymerase includes an amino acid sequence that is 95%identical to SEQ ID NO: 1.

In an aspect is provided a polymerase including an amino acid sequencethat is at least 80% identical to a continuous 500 amino acid sequencewithin SEQ ID NO: 1. In embodiments, the polymerase includes thefollowing amino acids: an alanine or serine at amino acid position 409or any amino acid that is functionally equivalent to the amino acidposition 409; a glycine or alanine at amino acid position 410 or anyamino acid that is functionally equivalent to the amino acid position410; and a proline, valine, isoleucine, glycine, or serine at amino acidposition 411 or any amino acid that is functionally equivalent to theamino acid position 411. In embodiments, the polymerase includes analanine at amino acid position 409 or any amino acid that isfunctionally equivalent to the amino acid position 409. In embodiments,the polymerase includes a serine at amino acid position 409 or any aminoacid that is functionally equivalent to the amino acid position 409. Inembodiments, the polymerase includes a glycine at amino acid position410 or any amino acid that is functionally equivalent to the amino acidposition 410. In embodiments, the polymerase includes an alanine atamino acid position 410 or any amino acid that is functionallyequivalent to the amino acid position 410. In embodiments, thepolymerase includes a proline at amino acid position 411 or any aminoacid that is functionally equivalent to the amino acid position 411. Inembodiments, the polymerase includes a valine at amino acid position 411or any amino acid that is functionally equivalent to the amino acidposition 411. In embodiments, the polymerase includes a serine at aminoacid position 411 or any amino acid that is functionally equivalent tothe amino acid position 411. In embodiments, the polymerase includes aglycine at amino acid position 411 or any amino acid that isfunctionally equivalent to the amino acid position 411. In embodiments,the polymerase includes an isoleucine at amino acid position 411 or anyamino acid that is functionally equivalent to the amino acid position411.

In embodiments, the polymerase includes an alanine at amino acidposition 141 and 143; a serine or alanine at amino acid position 409; analanine or glycine at amino acid position 410; and an proline, valine,isoleucine, or glycine at amino acid position 411. In embodiments, thepolymerase includes an alanine at amino acid position 141 and 143; aserine at amino acid position 409; an alanine at amino acid position410; and a proline at amino acid position 411. In embodiments, thepolymerase includes an alanine at amino acid position 141 and 143; aserine at amino acid position 409; an alanine at amino acid position410; and valine at amino acid position 411. In embodiments, thepolymerase includes an alanine at amino acid position 141 and 143; aserine at amino acid position 409; a glycine at amino acid position 410;and a proline at amino acid position 411. In embodiments, the polymeraseincludes an alanine at amino acid position 141 and 143; an alanine atamino acid position 409; an alanine at amino acid position 410; and anisoleucine at amino acid position 411. In embodiments, the polymeraseincludes an alanine at amino acid position 141 and 143; a serine atamino acid position 409; an alanine at amino acid position 410; and anisoleucine at amino acid position 411. In embodiments, the polymeraseincludes an alanine at amino acid position 141 and 143; a serine atamino acid position 409; an alanine at amino acid position 410; and aglycine at amino acid position 411. In embodiments, the polymeraseincludes an alanine at amino acid position 141 and 143; a serine atamino acid position 409; a glycine at amino acid position 410; and aproline at amino acid position 411. In embodiments, the amino acids atpositions 141, 143, 409, 410 and 411 (or any amino acid that isfunctionally equivalent to the amino acid position 141, 143, 409, 410,and 411) are alanine, alanine, serine, alanine, and proline,respectively. In embodiments, the amino acids at positions 141, 143,409, 410 and 411 (or any amino acid that is functionally equivalent tothe amino acid position 141, 143, 409, 410, and 411) are alanine,alanine, serine, alanine, and valine, respectively. In embodiments, theamino acids at positions 141, 143, 409, 410 and 411 (or any amino acidthat is functionally equivalent to the amino acid position 141, 143,409, 410, and 411) are alanine, alanine, serine, glycine, and proline,respectively. In embodiments, the amino acids at positions 141, 143,409, 410 and 411 (or any amino acid that is functionally equivalent tothe amino acid position 141, 143, 409, 410, and 411) are alanine,alanine, alanine, alanine, and valine, respectively. In embodiments, theamino acids at positions 141, 143, 409, 410 and 411 (or any amino acidthat is functionally equivalent to the amino acid position 141, 143,409, 410, and 411) are alanine, alanine, serine, alanine, andisoleucine, respectively. In embodiments, the amino acids at positions141, 143, 409, 410 and 411 (or any amino acid that is functionallyequivalent to the amino acid position 141, 143, 409, 410, and 411) arealanine, alanine, serine, alanine, and glycine, respectively. Inembodiments, the amino acids at positions 141, 143, 409, 410 and 411 (orany amino acid that is functionally equivalent to the amino acidposition 141, 143, 409, 410, and 411) are alanine, alanine, serine,glycine, and proline, respectively.

In embodiments, the polymerase further includes one or more mutationsselected from an alanine at at amino acid position 144; a glutamic acidat amino acid position 153; an alanine at amino acid position 215; analanine at amino acid position 215 and an alanine at amino acid position315; an alanine at position 315; a tryptophan at amino acid position477; an alanine at amino acid position 477; an alanine at amino acidposition 478; a tryptophan at position 477 and an alanine at position478; a serine at amino acid position 479; an alanine at position 477, analanine at position 478, and a serine at position 479; a valine at aminoacid position 486; a leucine at amino acid position 486; a serine atamino acid position 515; a leucine at amino acid position 522; anisoleucine at amino acid position 591; an alanine at amino acid position603, a leucine at amino acid position 640; a glutamic acid at amino acidposition 713; an alanine at amino acid position 714; an alanine at aminoacid position 719; an alanine at amino acid position 720; and an alanineat amino acid position 736.

In embodiments, the polymerase includes an alanine at amino acidpositions 129, 141, 143, and 486; a serine or alanine at amino acidposition 409; a glycine at amino acid position 410; and an isoleucine,proline, glycine, valine, or serine at amino acid position 411. Inembodiments, the polymerase includes an alanine at amino acid positions129, 141, 143, and 486; an alanine at amino acid position 409; a glycineat amino acid position 410; and isoleucine at amino acid position 411.In embodiments, the polymerase includes an alanine at amino acidpositions 129, 141, 143, and 486; an alanine at amino acid position 409;a glycine at amino acid position 410; and a proline at amino acidposition 411. In embodiments, the polymerase includes an alanine atamino acid positions 129, 141, 143, and 486; a serine at amino acidposition 409; a glycine at amino acid position 410; and an isoleucine atamino acid position 411. In embodiments, the polymerase includes analanine at amino acid positions 129, 141, 143, and 486; a serine atamino acid position 409; a glycine at amino acid position 410; and aproline at amino acid position 411. In embodiments, the polymeraseincludes an alanine at amino acid positions 129, 141, 143, and 486; analanine at amino acid position 409; a glycine at amino acid position410; and a glycine at amino acid position 411. In embodiments, thepolymerase includes an alanine at amino acid positions 129, 141, 143,and 486; an alanine at amino acid position 409; a glycine at amino acidposition 410; and a valine at amino acid position 411. In embodiments,the polymerase includes an alanine at amino acid positions 129, 141,143, and 486; a serine at amino acid position 409; a glycine at aminoacid position 410; and a serine at amino acid position 411. Inembodiments, the amino acids at positions 129, 141, 143, 409, 410, 411,and 486 (or any amino acid that is functionally equivalent to the aminoacid position 129, 141, 143, 409, 410, 411, and 486) are alanine,alanine, alanine, alanine, glycine, isoleucine, and alanine,respectively. In embodiments, the amino acids at positions 129, 141,143, 409, 410, 411, and 486 (or any amino acid that is functionallyequivalent to the amino acid position 129, 141, 143, 409, 410, 411, and486) are alanine, alanine, alanine, alanine, glycine, proline, andalanine, respectively. In embodiments, the amino acids at positions 129,141, 143, 409, 410, 411, and 486 (or any amino acid that is functionallyequivalent to the amino acid position 129, 141, 143, 409, 410, 411, and486) are alanine, alanine, alanine, serine, glycine, isoleucine, andalanine, respectively. In embodiments, the amino acids at positions 129,141, 143, 409, 410, 411, and 486 (or any amino acid that is functionallyequivalent to the amino acid position 129, 141, 143, 409, 410, 411, and486) are alanine, alanine, alanine, serine, glycine, proline, andalanine, respectively. In embodiments, the amino acids at positions 129,141, 143, 409, 410, 411, and 486 (or any amino acid that is functionallyequivalent to the amino acid position 129, 141, 143, 409, 410, 411, and486) are alanine, alanine, alanine, alanine, glycine, glycine, andalanine, respectively. In embodiments, the amino acids at positions 129,141, 143, 409, 410, 411, and 486 (or any amino acid that is functionallyequivalent to the amino acid position 129, 141, 143, 409, 410, 411, and486) are alanine, alanine, alanine, alanine, glycine, valine, andalanine, respectively. In embodiments, the amino acids at positions 129,141, 143, 409, 410, 411, and 486 (or any amino acid that is functionallyequivalent to the amino acid position 129, 141, 143, 409, 410, 411, and486) are alanine, alanine, alanine, serine, glycine, serine, andalanine, respectively.

In embodiments, the polymerase further includes one or more mutationsselected from an alanine at at amino acid position 144; a glutamic acidat amino acid position 153; an alanine at amino acid position 215; analanine at amino acid position 215 and an alanine at amino acid position315; an alanine at position 315; a tryptophan at amino acid position477; an alanine at amino acid position 477; an alanine at amino acidposition 478; a tryptophan at position 477 and an alanine at position478; a serine at amino acid position 479; an alanine at position 477, analanine at position 478, and a serine at position 479; a serine at aminoacid position 515; a leucine at amino acid position 522, an isoleucineat amino acid position 591; an alanine at amino acid position 603, aleucine at amino acid position 640; a glutamic acid at amino acidposition 713; an alanine at amino acid position 714; an alanine at aminoacid position 719; an alanine at amino acid position 720; and an alanineat amino acid position 736.

In embodiments, the polymerase includes an alanine at amino acidposition 141 and 143; a glutamic acid at amino acid position 153; aserine or alanine at amino acid position 409; an alanine or glycine atamino acid position 410; and an proline, valine, isoleucine, or glycineat amino acid position 411. In embodiments, the polymerase includes analanine at amino acid position 141 and 143; a glutamic acid at aminoacid position 153; a serine at amino acid position 409; an alanine atamino acid position 410; and a proline at position 411. In embodiments,the polymerase includes an alanine at amino acid position 141 and 143; aglutamic acid at amino acid position 153; a serine at amino acidposition 409; an alanine at amino acid position 410; and a valine atamino acid position 411. In embodiments, the polymerase includes analanine at amino acid position 141 and 143; a glutamic acid at aminoacid position 153; a serine at amino acid position 409; a glycine atamino acid position 410; and an isoleucine at amino acid position 411.In embodiments, the polymerase includes an alanine at amino acidposition 141 and 143; a glutamic acid at amino acid position 153; analanine at amino acid position 409; an alanine at amino acid position410; and a valine at amino acid position 411. In embodiments, thepolymerase includes an alanine at amino acid position 141 and 143; aglutamic acid at amino acid position 153; a serine at amino acidposition 409; an alanine at amino acid position 410; and an isoleucineat amino acid position 411. In embodiments, the polymerase includes analanine at amino acid position 141 and 143; a glutamic acid at aminoacid position 153; a serine at amino acid position 409; an alanine atamino acid position 410; and a glycine at amino acid position 411. Inembodiments, the polymerase includes an alanine at amino acid position141 and 143; a glutamic acid at amino acid position 153; a serine atamino acid position 409; a glycine at amino acid position 410; and aproline at amino acid position 411. In embodiments, the amino acids atpositions 141, 143, 153, 409, 410 and 411 (or any amino acid that isfunctionally equivalent to the amino acid position 141, 143, 153, 409,410, and 411) are alanine, alanine, glutamic acid, serine, alanine, andproline, respectively. In embodiments, the amino acids at positions 141,143, 153, 409, 410 and 411 (or any amino acid that is functionallyequivalent to the amino acid position 141, 143, 153, 409, 410, and 41)1are alanine, alanine, glutamic acid, serine, alanine, and valine,respectively. In embodiments, the amino acids at positions 141, 143,153, 409, 410 and 411 (or any amino acid that is functionally equivalentto the amino acid position 141, 143, 153, 409, 410, and 411) arealanine, alanine, glutamic acid serine, glycine, and isoleucine,respectively. In embodiments, the amino acids at positions 141, 143,153, 409, 410 and 411 (or any amino acid that is functionally equivalentto the amino acid position 141, 143, 153, 409, 410, and 411) arealanine, alanine, glutamic acid, alanine, alanine, and valine,respectively. In embodiments, the amino acids at positions 141, 143,153, 409, 410 and 411 (or any amino acid that is functionally equivalentto the amino acid position 141, 143, 153, 409, 410, and 411) arealanine, alanine, glutamic acid, serine, alanine, and isoleucine,respectively. In embodiments, the amino acids at positions 141, 143,153, 409, 410 and 411 (or any amino acid that is functionally equivalentto the amino acid position 141, 143, 153, 409, 410, and 411) arealanine, alanine, glutamic acid, serine, alanine, and glycine,respectively. In embodiments, the amino acids at positions 141, 143,153, 409, 410 and 411 (or any amino acid that is functionally equivalentto the amino acid position 141, 143, 153, 409, 410, and 411) arealanine, alanine, glutamic acid, serine, glycine, and proline,respectively.

In embodiments, the polymerase further includes one or more mutationsselected from an alanine at at amino acid position 144; an alanine atamino acid position 215; an alanine at amino acid position 215 and analanine at amino acid position 315; an alanine at position 315; atryptophan at amino acid position 477; an alanine at amino acid position477; an alanine at amino acid position 478; a tryptophan at position 477and an alanine at position 478; a serine at amino acid position 479; analanine at position 477, an alanine at position 478, and a serine atposition 479; a valine at amino acid position 486; a leucine at aminoacid position 486; a serine at amino acid position 515; a leucine atamino acid position 522, an isoleucine at amino acid position 591; analanine at amino acid position 603, a leucine at amino acid position640; a glutamic acid at amino acid position 713; an alanine at aminoacid position 714; an alanine at amino acid position 719; an alanine atamino acid position 720; and an alanine at amino acid position 736.

In embodiments, the polymerase includes an alanine at amino acidpositions 129, 141, 143, and 486; a glutamic acid at amino acid position153; a serine or alanine at amino acid position 409; a glycine at aminoacid position 410; and an isoleucine, proline, glycine, valine, orserine at amino acid position 411. In embodiments, the polymeraseincludes an alanine at amino acid positions 129, 141, 143, and 486; aglutamic acid at amino acid position 153; an alanine at amino acidposition 409; a glycine at amino acid position 410; and isoleucine atamino acid position 411. In embodiments, the polymerase includes analanine at amino acid positions 129, 141, 143, and 486; a glutamic acidat amino acid position 153; an alanine at amino acid position 409; aglycine at amino acid position 410; and a proline at amino acid position411. In embodiments, the polymerase includes an alanine at amino acidpositions 129, 141, 143, and 486; a glutamic acid at amino acid position153; a serine at amino acid position 409; a glycine at amino acidposition 410; and an isoleucine at amino acid position 411. Inembodiments, the polymerase includes an alanine at amino acid positions129, 141, 143, and 486; a glutamic acid at amino acid position 153; aserine at amino acid position 409; a glycine at amino acid position 410;and a proline at amino acid position 411. In embodiments, the polymeraseincludes an alanine at amino acid positions 129, 141, 143, and 486; aglutamic acid at amino acid position 153; an alanine at amino acidposition 409; a glycine at amino acid position 410; and a glycine atamino acid position 411. In embodiments, the polymerase includes analanine at amino acid positions 129, 141, 143, and 486; a glutamic acidat amino acid position 153; an alanine at amino acid position 409; aglycine at amino acid position 410; and a valine at amino acid position411. In embodiments, the polymerase includes an alanine at amino acidpositions 129, 141, 143, and 486; a glutamic acid at amino acid position153; a serine at amino acid position 409; a glycine at amino acidposition 410; and a serine at amino acid position 411. In embodiments,the amino acids at positions 129, 141, 143, 153, 409, 410, 411, and 486(or any amino acid that is functionally equivalent to the amino acidposition 129, 141, 143, 153, 409, 410, 411, and 486) are alanine,alanine, alanine, glutamic acid, alanine, glycine, isoleucine, andalanine, respectively. In embodiments, the amino acids at positions 129,141, 143, 153, 409, 410, 411, and 486 (or any amino acid that isfunctionally equivalent to the amino acid position 129, 141, 143, 153,409, 410, 411, and 486) are alanine, alanine, alanine, glutamic acid,alanine, glycine, proline, and alanine, respectively. In embodiments,the amino acids at positions 129, 141, 143, 153, 409, 410, 411, and 486(or any amino acid that is functionally equivalent to the amino acidposition 129, 141, 143, 154, 409, 410, 411, and 486) are alanine,alanine, alanine, glutamic acid, serine, glycine, isoleucine, andalanine, respectively. In embodiments, the amino acids at positions 129,141, 143, 153, 409, 410, 411, and 486 (or any amino acid that isfunctionally equivalent to the amino acid position 129, 141, 143, 153,409, 410, 411, and 486) are alanine, alanine, alanine, glutamic acid,serine, glycine, proline, and alanine, respectively. In embodiments, theamino acids at positions 129, 141, 143, 153, 409, 410, 411, and 486 (orany amino acid that is functionally equivalent to the amino acidposition 129, 141, 143, 153, 409, 410, 411, and 486) are alanine,alanine, alanine, glutamic acid, alanine, glycine, glycine, and alanine,respectively. In embodiments, the amino acids at positions 129, 141,143, 153, 409, 410, 411, and 486 (or any amino acid that is functionallyequivalent to the amino acid position 129, 141, 143, 153, 409, 410, 411,and 486) are alanine, alanine, alanine, glutamic acid, alanine, glycine,valine, and alanine, respectively. In embodiments, the amino acids atpositions 129, 141, 143, 153, 409, 410, 411, and 486 (or any amino acidthat is functionally equivalent to the amino acid position 129, 141,143, 153, 409, 410, 411, and 486) are alanine, alanine, alanine,glutamic acid, serine, glycine, serine, and alanine, respectively.

In embodiments, the polymerase further includes one or more mutationsselected from an alanine at at amino acid position 144; an alanine atamino acid position 215; an alanine at amino acid position 215 and analanine at amino acid position 315; an alanine at position 315; atryptophan at amino acid position 477; an alanine at amino acid position477; an alanine at amino acid position 478; a tryptophan at position 477and an alanine at position 478; a serine at amino acid position 479; analanine at position 477, an alanine at position 478, and a serine atposition 479; a serine at amino acid position 515; a leucine at aminoacid position 522, an isoleucine at amino acid position 591; an alanineat amino acid position 603, a leucine at amino acid position 640; aglutamic acid at amino acid position 713; an alanine at amino acidposition 714; an alanine at amino acid position 719; an alanine at aminoacid position 720; and an alanine at amino acid position 736.

In embodiments, the polymerase includes an amino acid substitutionmutation between position 129 and 736 of SEQ ID NO: 1, inclusive ofposition endpoints. In embodiments, the polymerase includes two aminoacid substitutions between position 129 and 736 of SEQ ID NO: 1,inclusive of position endpoints. In embodiments, the polymerase includesthree amino acid substitutions between position 129 and 736 of SEQ IDNO: 1, inclusive of position endpoints. In embodiments, the polymeraseincludes four amino acid substitutions between position 129 and 736 ofSEQ ID NO: 1, inclusive of position endpoints. In embodiments, thepolymerase includes five amino acid substitutions between position 129and 736 of SEQ ID NO: 1, inclusive of position endpoints.

In embodiments, the polymerase further includes an amino acidsubstitution mutation at positions 129, 141, 143, 153, 409, 410, and411. In embodiments, the polymerase further includes an amino acidsubstitution mutation at positions 129, 141, 143, 153, 409, 410 or 411.

In embodiments, the polymerase includes an alanine at amino acidposition 141; an alanine at amino acid position 143; a serine or alanineat amino acid position 409; an alanine or glycine at amino acid position410; and an valine, proline, isoleucine, or glycine at amino acidposition 411.

In embodiments, the polymerase includes an alanine at amino acidposition 129, an alanine at amino acid position 141; an alanine at aminoacid position 143; a serine or alanine at amino acid position 409; aglycine at amino acid position 410; and an isoleucine, proline, glycine,valine, or serine at amino acid position 411.

In embodiments, the polymerase includes an alanine at amino acidposition 141; an alanine at amino acid position 143; a glutamic acid atamino acid position 153; a serine or alanine at amino acid position 409;an alanine or glycine at amino acid position 410; and an valine,proline, isoleucine, or glycine at amino acid position 411.

In embodiments, the polymerase includes an alanine at amino acidposition 129, an alanine at amino acid position 141; an alanine at aminoacid position 143; a glutamic acid at amino acid position 153; a serineor alanine at amino acid position 409; a glycine at amino acid position410; and an isoleucine, proline, glycine, valine, or serine at aminoacid position 411.

In embodiments, the polymerase includes an amino acid substitution atposition 129. In embodiments, the amino acid substitution at position129 is an alanine substitution.

In embodiments, the polymerase includes an amino acid substitution atposition 141. In embodiments, the amino acid substitution at position141 is an alanine substitution.

In embodiments, the polymerase includes an amino acid substitution atposition 143. In embodiments, the amino acid substitution at position143 is an alanine substitution.

In embodiments, the polymerase includes an amino acid substitution atposition 144. In embodiments, the amino acid substitution at position144 is an alanine substitution.

In embodiments, the polymerase includes an amino acid substitution atposition 153. In embodiments, the amino acid substitution at position153 is a glutamic acid substitution. In embodiments, the amino acidsubstitution at position 153 is an aspartic acid substitution.

In embodiments, the polymerase includes an amino acid substitution atposition 215. In embodiments, the amino acid substitution at position215 is an alanine substitution.

In embodiments, the polymerase includes an amino acid substitution atposition 315. In embodiments, the amino acid substitution at position315 is an alanine substitution.

In embodiments, the polymerase includes an amino acid substitution atpositions 215 and 315. In embodiments, the amino acid substitution atpositions 215 and 315 are an alanine substitution.

In embodiments, the polymerase includes an amino acid substitution atposition 409. The amino acid substitution at position 409 may be aserine substitution or an alanine substitution. In embodiments, theamino acid substitution at position 409 is a serine substitution. Inembodiments, the amino acid substitution at position 409 is an alaninesubstitution.

In embodiments, the polymerase includes an amino acid substitution atposition 410. The amino acid substitution at position 410 may be aglycine substitution or an alanine substitution. In embodiments, theamino acid substitution at position 410 is a glycine substitution. Inembodiments, the amino acid substitution at position 410 is an alaninesubstitution.

In embodiments, the polymerase includes an amino acid substitution atposition 411. The amino acid substitution at position 411 may be anisoleucine substitution, a proline substitution, a glycine substitution,a valine substitution, or a serine substitution. In embodiments, theamino acid substitution at position 411 is an isoleucine substitution.In embodiments, the amino acid substitution at position 411 is a prolinesubstitution. In embodiments, the amino acid substitution at position411 is a glycine substitution. In embodiments, the amino acidsubstitution at position 411 is a valine substitution. In embodiments,the amino acid substitution at position 411 is a serine substitution.

In embodiments, the polymerase includes an amino acid substitution atposition 477. The amino acid substitution at position 477 may be atryptophan substitution or an alanine substitution. In embodiments, theamino acid substitution at position 477 is a tryptophan substitution. Inembodiments, the amino acid substitution at position 477 is an alaninesubstitution.

In embodiments, the polymerase includes an amino acid substitution atposition 478. In embodiments, the amino acid substitution at position478 is an alanine substitution.

In embodiments, the polymerase includes an amino acid substitution atposition 479. In embodiments, the amino acid substitution at position479 is a serine substitution.

In embodiments, the polymerase includes an amino acid substitution atpositions 477 and 478. In embodiments, the amino acid substitution atpositions 477 is a tryptophan substitution and at position 478 is analanine substitution. In embodiments, the amino acid substitution atpositions 477 and 478 are alanine substitutions.

In embodiments, the polymerase includes an amino acid substitution atpositions 477, 478 and 479. In embodiments, the amino acid substitutionat positions 477 is an alanine substation, at position 478 is an alaninesubstitution, and at position 479 is a serine substitution.

In embodiments, the polymerase includes an amino acid substitution atposition 486. In embodiments, the amino acid substitution at position486 is an alanine substitution. In embodiments, the amino acidsubstitution at position 486 is a valine substitution. In embodiments,the amino acid substitution at position 486 is a leucine substitution.In embodiments, the polymerase does not include an amino acidsubstitution at position 486, and amino acid position 486 is alanine.

In embodiments, the polymerase includes an amino acid substitution atposition 515. In embodiments, the amino acid substitution at position515 is a serine substitution.

In embodiments, the polymerase includes an amino acid substitution atposition 522. In embodiments, the amino acid substitution at position522 is a leucine substitution.

In embodiments, the polymerase includes an amino acid substitution atposition 591. In embodiments, the amino acid substitution at position591 is an isoleucine substitution.

In embodiments, the polymerase includes an amino acid substitution atposition 603. In embodiments, the amino acid substitution at position603 is an alanine substitution.

In embodiments, the polymerase includes an amino acid substitution atposition 640. In embodiments, the amino acid substitution at position640 is a leucine substitution.

In embodiments, the polymerase includes an amino acid substitution atposition 713. In embodiments, the amino acid substitution at position713 is a glutamic acid substitution.

In embodiments, the polymerase includes an amino acid substitution atposition 714. In embodiments, the amino acid substitution at position714 is an alanine substitution.

In embodiments, the polymerase includes an amino acid substitution atposition 719. In embodiments, the amino acid substitution at position719 is an alanine substitution.

In embodiments, the polymerase includes an amino acid substitution atposition 720. In embodiments, the amino acid substitution at position720 is an alanine substitution.

In embodiments, the polymerase includes an amino acid substitution atposition 736. In embodiments, the amino acid substitution at position736 is an alanine substitution.

In embodiments, the polymerase includes the following amino acidsubstitution mutations relative to SEQ ID NO: 1: D141A; E143A; L409S;Y410A; P411V. In embodiments, the polymerase includes the followingamino acid substitution mutations relative to SEQ ID NO: 1: D141A;E143A; L409S; Y410A; P411P. In embodiments, the polymerase includes thefollowing amino acid substitution mutations relative to SEQ ID NO: 1:D141A; E143A; L409S; Y410G; P411I. In embodiments, the polymeraseincludes the following amino acid substitution mutations relative to SEQID NO: 1: D141A; E143A; L409A; Y410A; P411V. In embodiments, thepolymerase includes the following amino acid substitution mutationsrelative to SEQ ID NO: 1: D141A; E143A; L409S; Y410A; P411I. Inembodiments, the polymerase includes the following amino acidsubstitution mutations relative to SEQ ID NO: 1: D141A; E143A; L409S;Y410A; P411G. In embodiments, the polymerase includes the followingamino acid substitution mutations relative to SEQ ID NO: 1: D141A;E143A; L409S; Y410G; and P411P.

In embodiments, the polymerase further includes one or more of thefollowing amino acid substitution mutations relative to SEQ ID NO: 1:T144A; G153E; D215A; D315A; D215A and D315A; T515S; I522L; T591I; K477W;K477A; K478A; L479S; K477W and K478A; K477A and K478A and L479S; A486V;A486L; K603A; A640L; K713E; R714A; E719A; E720A; or N736A.

In embodiments, the polymerase includes the following amino acidsubstitution mutations relative to SEQ ID NO: 1: M129A; D141A; E143A;L409A; Y410G; P411I; and A486A. In embodiments, the polymerase includesthe following amino acid substitution mutations relative to SEQ ID NO:1: M129A; D141A; E143A; L409A; Y410G; P411P; and A486A. In embodiments,the polymerase includes the following amino acid substitution mutationsrelative to SEQ ID NO: 1: M129A; D141A; E143A; L409S; Y410G; P411I;A486A. In embodiments, the polymerase includes the following amino acidsubstitution mutations relative to SEQ ID NO: 1: M129A; D141A; E143A;L409S; Y410G; P411P; and A486A. In embodiments, the polymerase includesthe following amino acid substitution mutations relative to SEQ ID NO:1: M129A; D141A; E143A; L409A; Y410G; P411G; and A486A. In embodiments,the polymerase includes the following amino acid substitution mutationsrelative to SEQ ID NO: 1: M129A; D141A; E143A; L409A; Y410G; P411V; andA486A. In embodiments, the polymerase includes the following amino acidsubstitution mutations relative to SEQ ID NO: 1: M129A; D141A; E143A;L409S; Y410G; P411S; and A486A.

In embodiments, the polymerase further includes one or more of thefollowing amino acid substitution mutations relative to SEQ ID NO: 1:T144A; G153E; D215A; D315A; D215A and D315A; T515S; I522L; T591I; K477W;K477A; K478A; L479S; K477W and K478A; K477A and K478A and L479S; K603A;A640L; K713E; R714A; E719A; E720A; or N736A.

In embodiments, the polymerase includes the following amino acidsubstitution mutations relative to SEQ ID NO: 1: D141A; E143A; G153E;L409S; Y410A; P411P. In embodiments, the polymerase includes thefollowing amino acid substitution mutations relative to SEQ ID NO: 1:D141A; E143A; G153E; L409S; Y410A; P411V. In embodiments, the polymeraseincludes the following amino acid substitution mutations relative to SEQID NO: 1: D141A; E143A; G153E; L409S; Y410G; P411I. In embodiments, thepolymerase includes the following amino acid substitution mutationsrelative to SEQ ID NO: 1: D141A; E143A; G153E; L409A; Y410A; P411V. Inembodiments, the polymerase includes the following amino acidsubstitution mutations relative to SEQ ID NO: 1:D141A; E143A; G153E;L409S; Y410A; P411I. In embodiments, the polymerase includes thefollowing amino acid substitution mutations relative to SEQ ID NO: 1:D141A; E143A; G153E; L409S; Y410A; P411G. In embodiments, the polymeraseincludes the following amino acid substitution mutations relative to SEQID NO: 1:D141A; E143A; G153E; L409S; Y410G; P411P

In embodiments, the polymerase further includes one or more of thefollowing amino acid substitution mutations relative to SEQ ID NO:1:T144A; D215A; D315A; D215A and D315A; T515S; I522L; T591I; K477W;K477A; K478A; L479S; K477W and K478A; K477A and K478A and L479S; K603A;A640L; K713E; R714A; E719A; E720A; or N736A.

In embodiments, the polymerase includes the following amino acidsubstitution mutations relative to SEQ ID NO: 1: M129A; D141A; E143A;G153E; L409A; Y410G; P411I; A486A. In embodiments, the polymeraseincludes the following amino acid substitution mutations relative to SEQID NO: 1: M129A; D141A; E143A; G153E; L409A; Y410G; P411P; A486A. Inembodiments, the polymerase includes the following amino acidsubstitution mutations relative to SEQ ID NO: 1: M129A; D141A; E143A;G153E; L409S; Y410G; P411I; A486A. In embodiments, the polymeraseincludes the following amino acid substitution mutations relative to SEQID NO: 1: M129A; D141A; E143A; G153E; L409S; Y410G; P411P; A486A. Inembodiments, the polymerase includes the following amino acidsubstitution mutations relative to SEQ ID NO: 1: M129A; D141A; E143A;G153E; L409A; Y410G; P411G; A486A. In embodiments, the polymeraseincludes the following amino acid substitution mutations relative to SEQID NO: 1: M129A; D141A; E143A; G153E; L409A; Y410G; P411V; A486A. Inembodiments, the polymerase includes the following amino acidsubstitution mutations relative to SEQ ID NO: 1: M129A; D141A; E143A;G153E; L409S; Y410G; P411S; A486A.

In embodiments, the polymerase further includes one or more of thefollowing amino acid substitution mutations relative to SEQ ID NO: 1:T144A; D215A; D315A; D215A and D315A; T515S; I522L; T591I; K477W; K477A;K478A; L479S; K477W and K478A; K477A and K478A and L479S; K603A; A640L;K713E; R714A; E719A; E720A; or N736A.

In an aspect, the polymerase (a synthetic or variant DNA polymerase)provided herein may have one or more amino acid substitution mutationsat position 129, 141, 143, 153, 409, 410, 411, and/or 486.

In embodiments, the polymerase is a polymerase described herein (e.g.,described in a claim, figure, or sequence listing). In embodiments, thepolymerase provided herein may have one or more amino acid substitutionmutations relative to the polymerase having the sequence described inSEQ ID NO: 1. In embodiments, the polymerase provided herein may haveone or more amino acid substitution mutations relative to the polymerasehaving the sequence described in SEQ ID NO: 21. In embodiments, thepolymerase provided herein may have one or more amino acid substitutionmutations relative to the polymerase having the sequence described inSEQ ID NO: 22. In embodiments, the polymerase provided herein may haveone or more amino acid substitution mutations relative to the polymerasehaving the sequence described in SEQ ID NO: 23. In embodiments, thepolymerase provided herein may have one or more amino acid substitutionmutations relative to the polymerase having the sequence described inSEQ ID NO: 24. In embodiments, the polymerase provided herein may haveone or more amino acid substitution mutations relative to the polymerasehaving the sequence described in SEQ ID NO: 25. In embodiments, thepolymerase provided herein may have one or more amino acid substitutionmutations relative to the polymerase having the sequence described inSEQ ID NO: 26. In embodiments, the polymerase provided herein may haveone or more amino acid substitution mutations relative to the polymerasehaving the sequence described in SEQ ID NO: 27. In embodiments, thepolymerase provided herein may have one or more amino acid substitutionmutations relative to the polymerase having the sequence described inSEQ ID NO: 28. In embodiments, the polymerase provided herein may haveone or more amino acid substitution mutations relative to the polymerasehaving the sequence described in SEQ ID NO: 29. In embodiments, thepolymerase provided herein may have one or more amino acid substitutionmutations relative to the polymerase having the sequence described inSEQ ID NO: 30.

In embodiments, the polymerase exhibits an increased rate ofincorporation of modified nucleotides, relative to a control (e.g.,wild-type P. horikoshii DNA polymerase (SEQ ID NO:1); wild-type P.abyssi DNA polymerase (SEQ ID NO:21); or a mutant polymerase (e.g., aDNA polymerase capable of incorporating modified nucleotides).). Inembodiments, the rate of incorporation is increased 1.1, 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2,4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6,5.7, 5.8, 5.9, or 6.0-fold relative to a control (e.g., wild-type P.horikoshii DNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNApolymerase (SEQ ID NO:21); or a mutant polymerase (e.g., a DNApolymerase capable of incorporating modified nucleotides). Inembodiments, the rate of incorporation is increased 1-fold, 2-fold,3-fold, 4-fold, 5-fold, or 6.0-fold relative to a control (e.g.,wild-type P. horikoshii DNA polymerase (SEQ ID NO:1); wild-type P.abyssi DNA polymerase (SEQ ID NO:21); or a mutant polymerase (e.g., aDNA polymerase capable of incorporating modified nucleotides). Inembodiments, the rate of incorporation of a modified nucleotide isincreased 1.1-fold relative to a control (e.g., wild-type P. horikoshiiDNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNA polymerase (SEQ IDNO:21); or a mutant polymerase (e.g., a DNA polymerase capable ofincorporating modified nucleotides). In embodiments, the rate ofincorporation of a modified nucleotide is increased 1.2-fold relative toa control (e.g., wild-type P. horikoshii DNA polymerase (SEQ ID NO:1);wild-type P. abyssi DNA polymerase (SEQ ID NO:21); or a mutantpolymerase (e.g., a DNA polymerase capable of incorporating modifiednucleotides). In embodiments, the rate of incorporation of a modifiednucleotide is increased 1.3-fold relative to a control (e.g., wild-typeP. horikoshii DNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNApolymerase (SEQ ID NO:21); or a mutant polymerase (e.g., a DNApolymerase capable of incorporating modified nucleotides). Inembodiments, the rate of incorporation of a modified nucleotide isincreased 1.4-fold relative to a control (e.g., wild-type P. horikoshiiDNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNA polymerase (SEQ IDNO:21); or a mutant polymerase (e.g., a DNA polymerase capable ofincorporating modified nucleotides). In embodiments, the rate ofincorporation of a modified nucleotide is increased 1.5-fold relative toa control (e.g., wild-type P. P. horikoshii or SG5000). In embodiments,the rate of incorporation of a modified nucleotide is increased 1.6-foldrelative to a control (e.g., wild-type P. horikoshii DNA polymerase (SEQID NO:1); wild-type P. abyssi DNA polymerase (SEQ ID NO:21); or a mutantpolymerase (e.g., a DNA polymerase capable of incorporating modifiednucleotides). In embodiments, the rate of incorporation of a modifiednucleotide is increased 1.7-fold relative to a control (e.g., wild-typeP. horikoshii DNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNApolymerase (SEQ ID NO:21); or a mutant polymerase (e.g., a DNApolymerase capable of incorporating modified nucleotides). Inembodiments, the rate of incorporation of a modified nucleotide isincreased 1.8-fold relative to a control (e.g., wild-type P. horikoshiiDNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNA polymerase (SEQ IDNO:21); or a mutant polymerase (e.g., a DNA polymerase capable ofincorporating modified nucleotides). In embodiments, the rate ofincorporation of a modified nucleotide is increased 1.9-fold relative toa control (e.g., wild-type P. horikoshii DNA polymerase (SEQ ID NO:1);wild-type P. abyssi DNA polymerase (SEQ ID NO:21); or a mutantpolymerase (e.g., a DNA polymerase capable of incorporating modifiednucleotides). In embodiments, the rate of incorporation of a modifiednucleotide is increased 2.0-fold relative to a control (e.g., wild-typeP. horikoshii DNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNApolymerase (SEQ ID NO:21); or a mutant polymerase (e.g., a DNApolymerase capable of incorporating modified nucleotides). Inembodiments, the rate of incorporation of a modified nucleotide isincreased 2.1-fold relative to a control (e.g., wild-type P. horikoshiiDNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNA polymerase (SEQ IDNO:21); or a mutant polymerase (e.g., a DNA polymerase capable ofincorporating modified nucleotides). In embodiments, the rate ofincorporation of a modified nucleotide is increased 2.2-fold relative toa control (e.g., wild-type P. horikoshii DNA polymerase (SEQ ID NO:1);wild-type P. abyssi DNA polymerase (SEQ ID NO:21); or a mutantpolymerase (e.g., a DNA polymerase capable of incorporating modifiednucleotides). In embodiments, the rate of incorporation of a modifiednucleotide is increased 2.3-fold relative to a control (e.g., wild-typeP. horikoshii DNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNApolymerase (SEQ ID NO:21); or a mutant polymerase (e.g., a DNApolymerase capable of incorporating modified nucleotides). Inembodiments, the rate of incorporation of a modified nucleotide isincreased 2.4-fold relative to a control (e.g., wild-type P. horikoshiiDNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNA polymerase (SEQ IDNO:21); or a mutant polymerase (e.g., a DNA polymerase capable ofincorporating modified nucleotides). In embodiments, the rate ofincorporation of a modified nucleotide is increased 2.5-fold relative toa control (e.g., wild-type P. horikoshii DNA polymerase (SEQ ID NO:1);wild-type P. abyssi DNA polymerase (SEQ ID NO:21); or a mutantpolymerase (e.g., a DNA polymerase capable of incorporating modifiednucleotides). In embodiments, the rate of incorporation of a modifiednucleotide is increased 2.6-fold relative to a control (e.g., wild-typeP. horikoshii DNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNApolymerase (SEQ ID NO:21); or a mutant polymerase (e.g., a DNApolymerase capable of incorporating modified nucleotides). Inembodiments, the rate of incorporation of a modified nucleotide isincreased 2.7-fold relative to a control (e.g., wild-type P. horikoshiiDNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNA polymerase (SEQ IDNO:21); or a mutant polymerase (e.g., a DNA polymerase capable ofincorporating modified nucleotides). In embodiments, the rate ofincorporation of a modified nucleotide is increased 2.8-fold relative toa control (e.g., wild-type P. horikoshii DNA polymerase (SEQ ID NO:1);wild-type P. abyssi DNA polymerase (SEQ ID NO:21); or a mutantpolymerase (e.g., a DNA polymerase capable of incorporating modifiednucleotides). In embodiments, the rate of incorporation of a modifiednucleotide is increased 2.9-fold relative to a control (e.g., wild-typeP. horikoshii DNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNApolymerase (SEQ ID NO:21); or a mutant polymerase (e.g., a DNApolymerase capable of incorporating modified nucleotides). Inembodiments, the rate of incorporation of a modified nucleotide isincreased 3.0-fold relative to a control (e.g., wild-type P. horikoshiiDNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNA polymerase (SEQ IDNO:21); or a mutant polymerase (e.g., a DNA polymerase capable ofincorporating modified nucleotides). In embodiments, the rate ofincorporation of a modified nucleotide is increased 3.1-fold relative toa control (e.g., wild-type P. horikoshii DNA polymerase (SEQ ID NO:1);wild-type P. abyssi DNA polymerase (SEQ ID NO:21); or a mutantpolymerase (e.g., a DNA polymerase capable of incorporating modifiednucleotides). In embodiments, the rate of incorporation of a modifiednucleotide is increased 3.2-fold relative to a control (e.g., wild-typeP. horikoshii DNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNApolymerase (SEQ ID NO:21); or a mutant polymerase (e.g., a DNApolymerase capable of incorporating modified nucleotides). Inembodiments, the rate of incorporation of a modified nucleotide isincreased 3.3-fold relative to a control (e.g., wild-type P. horikoshiiDNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNA polymerase (SEQ IDNO:21); or a mutant polymerase (e.g., a DNA polymerase capable ofincorporating modified nucleotides). In embodiments, the rate ofincorporation of a modified nucleotide is increased 3.4-fold relative toa control (e.g., wild-type P. horikoshii DNA polymerase (SEQ ID NO:1);wild-type P. abyssi DNA polymerase (SEQ ID NO:21); or a mutantpolymerase (e.g., a DNA polymerase capable of incorporating modifiednucleotides). In embodiments, the rate of incorporation of a modifiednucleotide is increased 3.5-fold relative to a control (e.g., wild-typeP. horikoshii DNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNApolymerase (SEQ ID NO:21); or a mutant polymerase (e.g., a DNApolymerase capable of incorporating modified nucleotides). Inembodiments, the rate of incorporation of a modified nucleotide isincreased 3.6-fold relative to a control (e.g., wild-type P. horikoshiiDNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNA polymerase (SEQ IDNO:21); or a mutant polymerase (e.g., a DNA polymerase capable ofincorporating modified nucleotides). In embodiments, the rate ofincorporation of a modified nucleotide is increased 3.7-fold relative toa control (e.g., wild-type P. horikoshii DNA polymerase (SEQ ID NO:1);wild-type P. abyssi DNA polymerase (SEQ ID NO:21); or a mutantpolymerase (e.g., a DNA polymerase capable of incorporating modifiednucleotides). In embodiments, the rate of incorporation of a modifiednucleotide is increased 3.8-fold relative to a control (e.g., wild-typeP. horikoshii DNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNApolymerase (SEQ ID NO:21); or a mutant polymerase (e.g., a DNApolymerase capable of incorporating modified nucleotides). Inembodiments, the rate of incorporation of a modified nucleotide isincreased 3.9-fold relative to a control (e.g., wild-type P. horikoshiiDNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNA polymerase (SEQ IDNO:21); or a mutant polymerase (e.g., a DNA polymerase capable ofincorporating modified nucleotides). In embodiments, the rate ofincorporation of a modified nucleotide is increased 4.0-fold relative toa control (e.g., wild-type P. horikoshii DNA polymerase (SEQ ID NO:1);wild-type P. abyssi DNA polymerase (SEQ ID NO:21); or a mutantpolymerase (e.g., a DNA polymerase capable of incorporating modifiednucleotides). In embodiments, the rate of incorporation of a modifiednucleotide is increased 4.1-fold relative to a control (e.g., wild-typeP. horikoshii DNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNApolymerase (SEQ ID NO:21); or a mutant polymerase (e.g., a DNApolymerase capable of incorporating modified nucleotides). Inembodiments, the rate of incorporation of a modified nucleotide isincreased 4.2-fold relative to a control (e.g., wild-type P. horikoshiiDNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNA polymerase (SEQ IDNO:21); or a mutant polymerase (e.g., a DNA polymerase capable ofincorporating modified nucleotides). In embodiments, the rate ofincorporation of a modified nucleotide is increased 4.3-fold relative toa control (e.g., wild-type P. horikoshii DNA polymerase (SEQ ID NO:1);wild-type P. abyssi DNA polymerase (SEQ ID NO:21); or a mutantpolymerase (e.g., a DNA polymerase capable of incorporating modifiednucleotides). In embodiments, the rate of incorporation of a modifiednucleotide is increased 4.4-fold relative to a control (e.g., wild-typeP. horikoshii r SG5000). In embodiments, the rate of incorporation of amodified nucleotide is increased 4.5-fold relative to a control (e.g.,wild-type P. horikoshii DNA polymerase (SEQ ID NO:1); wild-type P.abyssi DNA polymerase (SEQ ID NO:21); or a mutant polymerase (e.g., aDNA polymerase capable of incorporating modified nucleotides). Inembodiments, the rate of incorporation of a modified nucleotide isincreased 4.6-fold relative to a control (e.g., wild-type P. horikoshiiDNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNA polymerase (SEQ IDNO:21); or a mutant polymerase (e.g., a DNA polymerase capable ofincorporating modified nucleotides). In embodiments, the rate ofincorporation of a modified nucleotide is increased 4.7-fold relative toa control (e.g., wild-type P. horikoshii DNA polymerase (SEQ ID NO:1);wild-type P. abyssi DNA polymerase (SEQ ID NO:21); or a mutantpolymerase (e.g., a DNA polymerase capable of incorporating modifiednucleotides). In embodiments, the rate of incorporation of a modifiednucleotide is increased 4.8-fold relative to a control (e.g., wild-typeP. horikoshii DNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNApolymerase (SEQ ID NO:21); or a mutant polymerase (e.g., a DNApolymerase capable of incorporating modified nucleotides). Inembodiments, the rate of incorporation of a modified nucleotide isincreased 4.9-fold relative to a control (e.g., wild-type P. horikoshiiDNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNA polymerase (SEQ IDNO:21); or a mutant polymerase (e.g., a DNA polymerase capable ofincorporating modified nucleotides). In embodiments, the rate ofincorporation of a modified nucleotide is increased 5.0-fold relative toa control (e.g., wild-type P. horikoshii DNA polymerase (SEQ ID NO:1);wild-type P. abyssi DNA polymerase (SEQ ID NO:21); or a mutantpolymerase (e.g., a DNA polymerase capable of incorporating modifiednucleotides). In embodiments, the rate of incorporation of a modifiednucleotide is increased 5.1-fold relative to a control (e.g., wild-typeP. horikoshii DNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNApolymerase (SEQ ID NO:21); or a mutant polymerase (e.g., a DNApolymerase capable of incorporating modified nucleotides). Inembodiments, the rate of incorporation of a modified nucleotide isincreased 5.2-fold relative to a control (e.g., wild-type P. horikoshiiDNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNA polymerase (SEQ IDNO:21); or a mutant polymerase (e.g., a DNA polymerase capable ofincorporating modified nucleotides). In embodiments, the rate ofincorporation of a modified nucleotide is increased 5.3-fold relative toa control (e.g., wild-type P. horikoshii DNA polymerase (SEQ ID NO:1);wild-type P. abyssi DNA polymerase (SEQ ID NO:21); or a mutantpolymerase (e.g., a DNA polymerase capable of incorporating modifiednucleotides). In embodiments, the rate of incorporation of a modifiednucleotide is increased 5.4-fold relative to a control (e.g., wild-typeP. horikoshii DNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNApolymerase (SEQ ID NO:21); or a mutant polymerase (e.g., a DNApolymerase capable of incorporating modified nucleotides). Inembodiments, the rate of incorporation of a modified nucleotide isincreased 5.5-fold relative to a control (e.g., wild-type P. horikoshiiDNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNA polymerase (SEQ IDNO:21); or a mutant polymerase (e.g., a DNA polymerase capable ofincorporating modified nucleotides). In embodiments, the rate ofincorporation of a modified nucleotide is increased 5.6-fold relative toa control (e.g., wild-type P. horikoshii DNA polymerase (SEQ ID NO:1);wild-type P. abyssi DNA polymerase (SEQ ID NO:21); or a mutantpolymerase (e.g., a DNA polymerase capable of incorporating modifiednucleotides). In embodiments, the rate of incorporation of a modifiednucleotide is increased 5.7-fold relative to a control (e.g., wild-typeP. horikoshii DNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNApolymerase (SEQ ID NO:21); or a mutant polymerase (e.g., a DNApolymerase capable of incorporating modified nucleotides). Inembodiments, the rate of incorporation of a modified nucleotide isincreased 5.8-fold relative to a control (e.g., wild-type P. horikoshiiDNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNA polymerase (SEQ IDNO:21); or a mutant polymerase (e.g., a DNA polymerase capable ofincorporating modified nucleotides). In embodiments, the rate ofincorporation of a modified nucleotide is increased 5.9-fold relative toa control (e.g., wild-type P. horikoshii DNA polymerase (SEQ ID NO:1);wild-type P. abyssi DNA polymerase (SEQ ID NO:21); or a mutantpolymerase (e.g., a DNA polymerase capable of incorporating modifiednucleotides). In embodiments, the rate of incorporation of a modifiednucleotide is increased 6.0-fold relative to a control (e.g., wild-typeP. horikoshii DNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNApolymerase (SEQ ID NO:21); or a mutant polymerase (e.g., a DNApolymerase capable of incorporating modified nucleotides). Inembodiments, the rate of incorporation of a modified nucleotide isincreased 6.1-fold relative to a control (e.g., wild-type P. horikoshiiDNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNA polymerase (SEQ IDNO:21); or a mutant polymerase (e.g., a DNA polymerase capable ofincorporating modified nucleotides). In embodiments, the rate ofincorporation of a modified nucleotide is increased 6.2-fold relative toa control (e.g., wild-type P. horikoshii DNA polymerase (SEQ ID NO:1);wild-type P. abyssi DNA polymerase (SEQ ID NO:21); or a mutantpolymerase (e.g., a DNA polymerase capable of incorporating modifiednucleotides). In embodiments, the rate of incorporation of a modifiednucleotide is increased 6.3-fold relative to a control (e.g., wild-typeP. horikoshii DNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNApolymerase (SEQ ID NO:21); or a mutant polymerase (e.g., a DNApolymerase capable of incorporating modified nucleotides). Inembodiments, the rate of incorporation of a modified nucleotide isincreased 6.4-fold relative to a control (e.g., wild-type P. horikoshiiDNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNA polymerase (SEQ IDNO:21); or a mutant polymerase (e.g., a DNA polymerase capable ofincorporating modified nucleotides). In embodiments, the rate ofincorporation of a modified nucleotide is increased 6.5-fold relative toa control (e.g., wild-type P. horikoshii DNA polymerase (SEQ ID NO:1);wild-type P. abyssi DNA polymerase (SEQ ID NO:21); or a mutantpolymerase (e.g., a DNA polymerase capable of incorporating modifiednucleotides). In embodiments, the rate of incorporation of a modifiednucleotide is increased 6.6-fold relative to a control (e.g., wild-typeP. horikoshii DNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNApolymerase (SEQ ID NO:21); or a mutant polymerase (e.g., a DNApolymerase capable of incorporating modified nucleotides). Inembodiments, the rate of incorporation of a modified nucleotide isincreased 6.7-fold relative to a control (e.g., wild-type P. horikoshiiDNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNA polymerase (SEQ IDNO:21); or a mutant polymerase (e.g., a DNA polymerase capable ofincorporating modified nucleotides). In embodiments, the rate ofincorporation of a modified nucleotide is increased 6.8-fold relative toa control (e.g., wild-type P. horikoshii DNA polymerase (SEQ ID NO:1);wild-type P. abyssi DNA polymerase (SEQ ID NO:21); or a mutantpolymerase (e.g., a DNA polymerase capable of incorporating modifiednucleotides). In embodiments, the rate of incorporation of a modifiednucleotide is increased 6.9-fold relative to a control (e.g., wild-typeP. horikoshii DNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNApolymerase (SEQ ID NO:21); or a mutant polymerase (e.g., a DNApolymerase capable of incorporating modified nucleotides). Inembodiments, the rate of incorporation of a modified nucleotide isincreased about 2-fold relative to a control (e.g., wild-type P.horikoshii DNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNApolymerase (SEQ ID NO:21); or a mutant polymerase (e.g., a DNApolymerase capable of incorporating modified nucleotides). Inembodiments, the rate of incorporation of a modified nucleotide isincreased about 3-fold relative to a control (e.g., wild-type P.horikoshii DNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNApolymerase (SEQ ID NO:21); or a mutant polymerase (e.g., a DNApolymerase capable of incorporating modified nucleotides). Inembodiments, the rate of incorporation of a modified nucleotide isincreased about 4-fold relative to a control (e.g., wild-type P.horikoshii DNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNApolymerase (SEQ ID NO:21); or a mutant polymerase (e.g., a DNApolymerase capable of incorporating modified nucleotides). Inembodiments, the rate of incorporation of a modified nucleotide isincreased about 5-fold relative to a control (e.g., wild-type P.horikoshii DNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNApolymerase (SEQ ID NO:21); or a mutant polymerase (e.g., a DNApolymerase capable of incorporating modified nucleotides). Inembodiments, the rate of incorporation of a modified nucleotide isincreased about 6-fold relative to a control (e.g., wild-type P.horikoshii DNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNApolymerase (SEQ ID NO:21); or a mutant polymerase (e.g., a DNApolymerase capable of incorporating modified nucleotides). Inembodiments, the rate of incorporation of a modified nucleotide isincreased about 7-fold relative to a control (e.g., wild-type P.horikoshii DNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNApolymerase (SEQ ID NO:21); or a mutant polymerase (e.g., a DNApolymerase capable of incorporating modified nucleotides). Inembodiments, the rate of incorporation of a modified nucleotide isincreased about 8-fold relative to a control (e.g., wild-type P.horikoshii DNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNApolymerase (SEQ ID NO:21); or a mutant polymerase (e.g., a DNApolymerase capable of incorporating modified nucleotides). Inembodiments, the rate of incorporation of a modified nucleotide isincreased about 9-fold relative to a control (e.g., wild-type P.horikoshii DNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNApolymerase (SEQ ID NO:21); or a mutant polymerase (e.g., a DNApolymerase capable of incorporating modified nucleotides). Inembodiments, the rate of incorporation of a modified nucleotide isincreased about 10-fold relative to a control (e.g., wild-type P.horikoshii DNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNApolymerase (SEQ ID NO:21); or a mutant polymerase (e.g., a DNApolymerase capable of incorporating modified nucleotides). Inembodiments, the rate of incorporation of a modified nucleotide isincreased 2-fold relative to a control (e.g., wild-type P. horikoshiiDNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNA polymerase (SEQ IDNO:21); or a mutant polymerase (e.g., a DNA polymerase capable ofincorporating modified nucleotides). In embodiments, the rate ofincorporation of a modified nucleotide is increased 3-fold relative to acontrol (e.g., wild-type P. horikoshii DNA polymerase (SEQ ID NO:1);wild-type P. abyssi DNA polymerase (SEQ ID NO:21); or a mutantpolymerase (e.g., a DNA polymerase capable of incorporating modifiednucleotides). In embodiments, the rate of incorporation of a modifiednucleotide is increased 4-fold relative to a control (e.g., wild-type P.horikoshii DNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNApolymerase (SEQ ID NO:21); or a mutant polymerase (e.g., a DNApolymerase capable of incorporating modified nucleotides). Inembodiments, the rate of incorporation of a modified nucleotide isincreased 5-fold relative to a control (e.g., wild-type P. horikoshiiDNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNA polymerase (SEQ IDNO:21); or a mutant polymerase (e.g., a DNA polymerase capable ofincorporating modified nucleotides). In embodiments, the rate ofincorporation of a modified nucleotide is increased 6-fold relative to acontrol (e.g., wild-type P. horikoshii DNA polymerase (SEQ ID NO:1);wild-type P. abyssi DNA polymerase (SEQ ID NO:21); or a mutantpolymerase (e.g., a DNA polymerase capable of incorporating modifiednucleotides). In embodiments, the rate of incorporation of a modifiednucleotide is increased 7-fold relative to a control (e.g., wild-type P.horikoshii DNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNApolymerase (SEQ ID NO:21); or a mutant polymerase (e.g., a DNApolymerase capable of incorporating modified nucleotides). Inembodiments, the rate of incorporation of a modified nucleotide isincreased 8-fold relative to a control (e.g., wild-type P. horikoshiiDNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNA polymerase (SEQ IDNO:21); or a mutant polymerase (e.g., a DNA polymerase capable ofincorporating modified nucleotides). In embodiments, the rate ofincorporation of a modified nucleotide is increased 9-fold relative to acontrol (e.g., wild-type P. horikoshii DNA polymerase (SEQ ID NO:1);wild-type P. abyssi DNA polymerase (SEQ ID NO:21); or a mutantpolymerase (e.g., a DNA polymerase capable of incorporating modifiednucleotides). In embodiments, the rate of incorporation of a modifiednucleotide is increased 10-fold relative to a control (e.g., wild-typeP. horikoshii DNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNApolymerase (SEQ ID NO:21); or a mutant polymerase (e.g., a DNApolymerase capable of incorporating modified nucleotides). Inembodiments, the rate of incorporation of a modified nucleotide isincreased 15-fold relative to a control (e.g., wild-type P. horikoshiiDNA polymerase (SEQ ID NO:1); wild-type P. abyssi DNA polymerase (SEQ IDNO:21); or a mutant polymerase (e.g., a DNA polymerase capable ofincorporating modified nucleotides). In embodiments, the rate ofincorporation of a modified nucleotide is increased 20-fold relative toa control (e.g., wild-type P. horikoshii DNA polymerase (SEQ ID NO:1);wild-type P. abyssi DNA polymerase (SEQ ID NO:21); or a mutantpolymerase (e.g., a DNA polymerase capable of incorporating modifiednucleotides). In embodiments, the control is SEQ ID NO: 31. Inembodiments, the control is a mutant polymerase (e.g., Thermococcus sp.9 degrees N-7, VentR®, VentR® (exo-), Deep VentR™, Deep VentR™ (exo-),Taq 9° N™, Phusion®, Long Amp® Taq, Long Amp® Hot Start Taq, One Taq®,and Q5™ or mutant thereof). Vent and Deep Vent are commercial names usedfor family B DNA polymerases isolated from the hyperthermophilicarchaeon Thermococcus litoralis. 9° N polymerase was also identifiedfrom Thermococcus sp.

In embodiments, the polymerase includes an amino acid sequence that isat least 95% identical to a continuous 500 amino acid sequence withinSEQ ID NO: 1. The polymerase includes substitution mutations atpositions 141 and 143 of SEQ ID NO: 1. The polymerase further includesat least one amino acid substitution mutation at a position selectedfrom positions 409, 410, or 411 of SEQ ID NO: 1.

In embodiments, the polymerase includes an amino acid sequence that isat least 95% identical to a continuous 500 amino acid sequence withinSEQ ID NO: 1. The polymerase includes substitution mutations atpositions 129, 141, 143, and 486 of SEQ ID NO: 1. The polymerase furtherincludes at least one amino acid substitution mutation at a positionselected from positions 409, 410, or 411 of SEQ ID NO: 1.

In embodiments, the polymerase includes an amino acid sequence that isat least 95% identical to a continuous 500 amino acid sequence withinSEQ ID NO: 1. The polymerase includes substitution mutations atpositions 141, 143, and 153 of SEQ ID NO: 1. The polymerase furtherincludes at least one amino acid substitution mutation at a positionselected from positions 409, 410, or 411 of SEQ ID NO: 1.

In embodiments, the polymerase includes an amino acid sequence that isat least 95% identical to a continuous 500 amino acid sequence withinSEQ ID NO: 1. The polymerase includes substitution mutations atpositions 129, 141, 143, 153, or 486 of SEQ ID NO: 1. The polymerasefurther includes at least one amino acid substitution mutation at aposition selected from positions 409, 410, or 411 of SEQ ID NO: 1.

In embodiments, the polymerase includes an amino acid sequence that isat least 85%, at least 90%, or at least 95% identical to a continuous500 amino acid sequence within SEQ ID NO: 1. In embodiments, thepolymerase includes an amino acid sequence that is at least 80%, atleast 85%, at least 90%, or at least 95% identical to a continuous 600amino acid sequence within SEQ ID NO: 1. In embodiments, the polymeraseincludes an amino acid sequence that is at least 80%, at least 85%, atleast 90%, or at least 95% identical to a continuous 700 amino acidsequence within SEQ ID NO: 1. In embodiments, the polymerase includes anamino acid sequence that is at least 85% identical to a continuous 500amino acid sequence within SEQ ID NO: 1. In embodiments, the polymeraseincludes an amino acid sequence that is at least 90% identical to acontinuous 500 amino acid sequence within SEQ ID NO: 1. In embodiments,the polymerase includes an amino acid sequence that is at least 95%identical to a continuous 500 amino acid sequence within SEQ ID NO: 1.In embodiments, the polymerase includes an amino acid sequence that isat least 85% identical to a continuous 600 amino acid sequence withinSEQ ID NO: 1. In embodiments, the polymerase includes an amino acidsequence that is at least 90% identical to a continuous 600 amino acidsequence within SEQ ID NO: 1. In embodiments, the polymerase includes anamino acid sequence that is at least 95% identical to a continuous 600amino acid sequence within SEQ ID NO: 1. In embodiments, the polymeraseincludes an amino acid sequence that is at least 85% identical to acontinuous 700 amino acid sequence within SEQ ID NO: 1. In embodiments,the polymerase includes an amino acid sequence that is at least 90%identical to a continuous 700 amino acid sequence within SEQ ID NO: 1.In embodiments, the polymerase includes an amino acid sequence that isat least 95% identical to a continuous 700 amino acid sequence withinSEQ ID NO: 1.

In embodiments, the polymerase includes alanine substitution mutationsat positions 141 and 143 of SEQ ID NO: 1. In embodiments, the polymerasefurther includes at least one amino acid substitution mutation at aposition selected from positions 129, 153, 409, 410, 411 and/or 486 ofSEQ ID NO: 1. In embodiments, the polymerase further includes at leastone amino acid substitution at a position selected from an alanine atposition 129; a glutamic acid at position 153; a serine or an alanine atposition 409; a glycine or alanine at position 410; a proline, valine,isoleucine, glycine, or serine at position 411, and an alanine 486.

The polymerase (a synthetic or variant DNA polymerase) may have asequence that is at least 60%, at least 65%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, or at least 95% identical tothe wild-type sequence of P. horikoshii family B DNA polymerase of SEQID NO: 1. In embodiments, the polymerase (a synthetic or variant DNApolymerase) may have a sequence that is 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, or 69% identical to the wild-type sequence of P.horikoshii family B DNA polymerase of SEQ ID NO: 1. The polymerase (asynthetic or variant DNA polymerase) may have a sequence that is 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79% identical to thewild-type sequence of P. horikoshii family B DNA polymerase of SEQ IDNO: 1. The polymerase (a synthetic or variant DNA polymerase) may have asequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%identical to the wild-type sequence of P. horikoshii family B DNApolymerase of SEQ ID NO: 1. The polymerase (a synthetic or variant DNApolymerase) may have a sequence that is 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to the wild-type sequence of P.horikoshii family B DNA polymerase of SEQ ID NO: 1

The polymerase (a synthetic or variant DNA polymerase) may have asequence that is identical to a continuous 500, 600, or 700 continuousamino acids of SEQ ID NO: 1. In embodiments, the polymerase (a syntheticor variant DNA polymerase) may have a sequence that is identical to acontinuous 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555,560, 565, 570, 575, 580, 585, 590, or 595 continuous amino acids of SEQID NO: 1. In embodiments, the polymerase (a synthetic or variant DNApolymerase) may have a sequence that is identical to a continuous 600,605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670,675, 680, 685, 690, or 695 continuous amino acids of SEQ ID NO: 1.

Examples of mutations giving rise to an exo-/exo-variants includemutations at positions in a parent polymerase corresponding to positionsin SEQ ID NO: 1 identified as follows: 141 and 143.

In embodiments, mutations may include substitution of the amino acid inthe parent amino acid sequences with an amino acid, which is not theparent amino acid. In embodiments, the mutations may result inconservative amino acid changes. In embodiments, non-polar amino acidsmay be converted into polar amino acids (threonine, asparagine,glutamine, cysteine, tyrosine, aspartic acid, glutamic acid orhistidine) or the parent amino acid may be changed to an alanine.

Alternatively, mutations may be randomly generated within the variousmotifs (within or outside the highly conserved sequences described)using standard techniques known in the art and the resultant enzymes canbe tested using the sensitive assays described in the Examples todetermine whether they have substantially no exonuclease activity.

In embodiments, the polymerase does not comprise the followingmutations: (L409S); (L409Q); (L409Y); or (L409F); (Y410G); (Y410A); or(Y410S); and (P411S); (P411I); (P411C); (P411A). In embodiments, thepolymerase does not comprise L409S; Y410G; and P411I. In embodiments,the polymerase does not comprise L409S; Y410A; and P411I. Inembodiments, the polymerase does not comprise L409S; Y410G; and P411S.In embodiments, the polymerase does not comprise L409S; Y410A; andP411S. In embodiments, the polymerase is not a wild type enzyme. Inembodiments, the polymerase is a synthetic polymerase.

Functionally equivalent, positionally equivalent and homologous aminoacids within the wild type amino acid sequences of two differentpolymerases do not necessarily have to be the same type of amino acidresidue, although functionally equivalent, positionally equivalent andhomologous amino acids are commonly conserved. By way of example, themotif A region of 9° N polymerase has the sequence LYP, the functionallyhomologous region of Vent™ polymerase also has sequence LYP. In the caseof these two polymerases the homologous amino acid sequences areidentical, however homologous regions in other polymerases may havedifferent amino acid sequence.

In embodiments, the polymerase is capable of incorporating an A-Term,S-Term, or i-term (e.g., i-term1 or iterm2) reversible terminatormoiety. In embodiments, the polymerase incorporates an A-Term, S-Term,or i-term (e.g., i-term1 or iterm2) reversible terminator moiety. A-Termrefers to azide-containing terminators (Guo J, et al. PNAS 2008); forexample having the formula:

S-Term refers to sulfide-containing terminators (WO 2017/058953); forexample having the formula

Wherein R″ is unsubstituted C₁-C₄ alkyl. The i-term probe refers to anisomeric reversible terminator For example, an i-term probe has theformula:

wherein R^(A) and R^(B) are hydrogen or alkyl, wherein at least one ofR^(A) or R^(B) are hydrogen to yield a steroisomeric probe, and R^(C) isthe remainder of the reversible terminator.

Certain mutations in the polymerase favor the incorporation of oneisomer, thus creating optimized polymerases for a unique class ofreversible terminators (i.e., isomeric reversible terminators). Inembodiments, the polymerase exhibits isomeric preference (i.e. itincorporates a modified nucleotide of one isomer (e.g., i-term1) at afaster rate than it incorporates a modified nucleotide of a differentisomer (e.g., i-term2).

In embodiments, the nucleotide is

wherein Base is a Base as described herein, R³ is —OH, monophosphate, orpolyphosphate or a nucleic acid, and R′ is a reversible terminatorhaving the formula:

wherein R^(A) and R^(B) are hydrogen or alkyl and R^(C) is the remainderof the reversible terminator. In embodiments, R^(A) is methyl, R^(B) ishydrogen, and R^(C) is the remainder of the reversible terminatormoiety. In embodiments, R′ has the formula

wherein R^(C) is the remainder of the reversible terminator moiety. Inembodiments, i-term1 has the formula

and i-term 2 has the formula:

wherein R^(C) is the remainder of the reversible terminator moiety(e.g., —N₃ or —SS-alkyl).

In embodiments, the polymerase is SE-1, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-2, having themutations as described in Table 6. In embodiments, the polymerase isSE-3, having the mutations as described in Table 6. In embodiments, thepolymerase is SE-4, having the mutations as described in Table 6. Inembodiments, the polymerase is SE-5, having the mutations as describedin Table 6. In embodiments, the polymerase is SE-6, having the mutationsas described in Table 6. In embodiments, the polymerase is SE-7, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-8, having the mutations as described in Table 6. In embodiments, thepolymerase is SE-9, having the mutations as described in Table 6. Inembodiments, the polymerase is SE-10, having the mutations as describedin Table 6. In embodiments, the polymerase is SE-11, having themutations as described in Table 6. In embodiments, the polymerase isSE-12, having the mutations as described in Table 6. In embodiments, thepolymerase is SE-13, having the mutations as described in Table 6. Inembodiments, the polymerase is SE-14, having the mutations as describedin Table 6. In embodiments, the polymerase is SE-15, having themutations as described in Table 6. In embodiments, the polymerase isSE-16, having the mutations as described in Table 6. In embodiments, thepolymerase is SE-17, having the mutations as described in Table 6. Inembodiments, the polymerase is SE-18, having the mutations as describedin Table 6. In embodiments, the polymerase is SE-19, having themutations as described in Table 6. In embodiments, the polymerase isSE-20, having the mutations as described in Table 6. In embodiments, thepolymerase is SE-21, having the mutations as described in Table 6. Inembodiments, the polymerase is SE-22, having the mutations as describedin Table 6. In embodiments, the polymerase is SE-23, having themutations as described in Table 6. In embodiments, the polymerase isSE-24, having the mutations as described in Table 6. In embodiments, thepolymerase is SE-25, having the mutations as described in Table 6. Inembodiments, the polymerase is SE-26, having the mutations as describedin Table 6. In embodiments, the polymerase is SE-27, having themutations as described in Table 6. In embodiments, the polymerase isSE-28, having the mutations as described in Table 6. In embodiments, thepolymerase is SE-29, having the mutations as described in Table 6. Inembodiments, the polymerase is SE-30, having the mutations as describedin Table 6. In embodiments, the polymerase is SE-31, having themutations as described in Table 6. In embodiments, the polymerase isSE-32, having the mutations as described in Table 6. In embodiments, thepolymerase is SE-33, having the mutations as described in Table 6. Inembodiments, the polymerase is SE-35, having the mutations as describedin Table 6. In embodiments, the polymerase is SE-36, having themutations as described in Table 6. In embodiments, the polymerase isSE-37, having the mutations as described in Table 6. In embodiments, thepolymerase is SE-38, having the mutations as described in Table 6. Inembodiments, the polymerase is SE-39, having the mutations as describedin Table 6. In embodiments, the polymerase is SE-40, having themutations as described in Table 6. In embodiments, the polymerase isSE-41, having the mutations as described in Table 6. In embodiments, thepolymerase is SE-42, having the mutations as described in Table 6. Inembodiments, the polymerase is SE-43, having the mutations as describedin Table 6. In embodiments, the polymerase is SE-44, having themutations as described in Table 6. In embodiments, the polymerase isSE-45, having the mutations as described in Table 6. In embodiments, thepolymerase is SE-46, having the mutations as described in Table 6. Inembodiments, the polymerase is SE-47, having the mutations as describedin Table 6. In embodiments, the polymerase is SE-48, having themutations as described in Table 6. In embodiments, the polymerase isSE-49, having the mutations as described in Table 6. In embodiments, thepolymerase is SE-50, having the mutations as described in Table 6. Inembodiments, the polymerase is SE-51, having the mutations as describedin Table 6. In embodiments, the polymerase is SE-52, having themutations as described in Table 6. In embodiments, the polymerase isSE-53, having the mutations as described in Table 6. In embodiments, thepolymerase is SE-54, having the mutations as described in Table 6. Inembodiments, the polymerase is SE-55, having the mutations as describedin Table 6. In embodiments, the polymerase is SE-56, having themutations as described in Table 6. In embodiments, the polymerase isSE-57, having the mutations as described in Table 6. In embodiments, thepolymerase is SE-58, having the mutations as described in Table 6. Inembodiments, the polymerase is SE-59, having the mutations as describedin Table 6. In embodiments, the polymerase is SE-60, having themutations as described in Table 6. In embodiments, the polymerase isSE-61, having the mutations as described in Table 6. In embodiments, thepolymerase is SE-62, having the mutations as described in Table 6. Inembodiments, the polymerase is SE-63, having the mutations as describedin Table 6. In embodiments, the polymerase is SE-64, having themutations as described in Table 6. In embodiments, the polymerase isSE-65, having the mutations as described in Table 6. In embodiments, thepolymerase is SE-66, having the mutations as described in Table 6. Inembodiments, the polymerase is SE-67, having the mutations as describedin Table 6. In embodiments, the polymerase is SE-68, having themutations as described in Table 6. In embodiments, the polymerase isSE-69, having the mutations as described in Table 6. In embodiments, thepolymerase is SE-70, having the mutations as described in Table 6. Inembodiments, the polymerase is SE-71, having the mutations as describedin Table 6. In embodiments, the polymerase is SE-72, having themutations as described in Table 6. In embodiments, the polymerase isSE-73, having the mutations as described in Table 6. In embodiments, thepolymerase is SE-74, having the mutations as described in Table 6. Inembodiments, the polymerase is SE-75, having the mutations as describedin Table 6. In embodiments, the polymerase is SE-76, having themutations as described in Table 6. In embodiments, the polymerase isSE-77, having the mutations as described in Table 6. In embodiments, thepolymerase is SE-78, having the mutations as described in Table 6. Inembodiments, the polymerase is SE-79, having the mutations as describedin Table 6. In embodiments, the polymerase is SE-80, having themutations as described in Table 6. In embodiments, the polymerase isSE-81, having the mutations as described in Table 6. In embodiments, thepolymerase is SE-82, having the mutations as described in Table 6. Inembodiments, the polymerase is SE-83, having the mutations as describedin Table 6. In embodiments, the polymerase is SE-84, having themutations as described in Table 6. In embodiments, the polymerase isSE-85, having the mutations as described in Table 6. In embodiments, thepolymerase is SE-86, having the mutations as described in Table 6. Inembodiments, the polymerase is SE-87, having the mutations as describedin Table 6. In embodiments, the polymerase is SE-88, having themutations as described in Table 6. In embodiments, the polymerase isSE-89, having the mutations as described in Table 6. In embodiments, thepolymerase is SE-90, having the mutations as described in Table 6. Inembodiments, the polymerase is SE-91, having the mutations as describedin Table 6. In embodiments, the polymerase is SE-92, having themutations as described in Table 6. In embodiments, the polymerase isSE-93, having the mutations as described in Table 6. In embodiments, thepolymerase is SE-94, having the mutations as described in Table 6. Inembodiments, the polymerase is SE-95, having the mutations as describedin Table 6. In embodiments, the polymerase is SE-96, having themutations as described in Table 6. In embodiments, the polymerase isSE-97, having the mutations as described in Table 6. In embodiments, thepolymerase is SE-98, having the mutations as described in Table 6. Inembodiments, the polymerase is SE-99, having the mutations as describedin Table 6. In embodiments, the polymerase is SE-100, having themutations as described in Table 6. In embodiments, the polymerase isSE-101, having the mutations as described in Table 6. In embodiments,the polymerase is SE-102, having the mutations as described in Table 6.In embodiments, the polymerase is SE-103, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-104, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-105, having the mutations as described in Table 6. In embodiments,the polymerase is SE-106, having the mutations as described in Table 6.In embodiments, the polymerase is SE-107, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-108, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-109, having the mutations as described in Table 6. In embodiments,the polymerase is SE-110, having the mutations as described in Table 6.In embodiments, the polymerase is SE-111, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-112, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-113, having the mutations as described in Table 6. In embodiments,the polymerase is SE-114, having the mutations as described in Table 6.In embodiments, the polymerase is SE-115, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-116, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-117, having the mutations as described in Table 6. In embodiments,the polymerase is SE-118, having the mutations as described in Table 6.In embodiments, the polymerase is SE-119, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-120, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-121, having the mutations as described in Table 6. In embodiments,the polymerase is SE-122, having the mutations as described in Table 6.In embodiments, the polymerase is SE-123, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-124, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-125, having the mutations as described in Table 6. In embodiments,the polymerase is SE-126, having the mutations as described in Table 6.In embodiments, the polymerase is SE-127, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-128, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-129, having the mutations as described in Table 6. In embodiments,the polymerase is SE-130, having the mutations as described in Table 6.In embodiments, the polymerase is SE-131, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-132, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-133, having the mutations as described in Table 6. In embodiments,the polymerase is SE-134, having the mutations as described in Table 6.In embodiments, the polymerase is SE-135, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-136, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-137, having the mutations as described in Table 6. In embodiments,the polymerase is SE-138, having the mutations as described in Table 6.In embodiments, the polymerase is SE-139, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-140, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-141, having the mutations as described in Table 6. In embodiments,the polymerase is SE-142, having the mutations as described in Table 6.In embodiments, the polymerase is SE-143, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-145, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-146, having the mutations as described in Table 6. In embodiments,the polymerase is SE-147, having the mutations as described in Table 6.In embodiments, the polymerase is SE-148, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-149, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-150, having the mutations as described in Table 6. In embodiments,the polymerase is SE-151, having the mutations as described in Table 6.In embodiments, the polymerase is SE-152, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-153, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-154, having the mutations as described in Table 6. In embodiments,the polymerase is SE-155, having the mutations as described in Table 6.In embodiments, the polymerase is SE-156, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-157, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-158, having the mutations as described in Table 6. In embodiments,the polymerase is SE-159, having the mutations as described in Table 6.In embodiments, the polymerase is SE-160, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-161, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-162, having the mutations as described in Table 6. In embodiments,the polymerase is SE-163, having the mutations as described in Table 6.In embodiments, the polymerase is SE-164, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-165, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-166, having the mutations as described in Table 6. In embodiments,the polymerase is SE-167, having the mutations as described in Table 6.In embodiments, the polymerase is SE-168, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-169, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-170, having the mutations as described in Table 6. In embodiments,the polymerase is SE-171, having the mutations as described in Table 6.In embodiments, the polymerase is SE-172, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-173, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-174, having the mutations as described in Table 6. In embodiments,the polymerase is SE-175, having the mutations as described in Table 6.In embodiments, the polymerase is SE-176, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-177, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-178, having the mutations as described in Table 6. In embodiments,the polymerase is SE-179, having the mutations as described in Table 6.In embodiments, the polymerase is SE-180, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-181, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-182, having the mutations as described in Table 6. In embodiments,the polymerase is SE-183, having the mutations as described in Table 6.In embodiments, the polymerase is SE-184, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-185, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-186, having the mutations as described in Table 6. In embodiments,the polymerase is SE-187, having the mutations as described in Table 6.In embodiments, the polymerase is SE-188, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-189, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-190, having the mutations as described in Table 6. In embodiments,the polymerase is SE-191, having the mutations as described in Table 6.In embodiments, the polymerase is SE-192, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-193, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-194, having the mutations as described in Table 6. In embodiments,the polymerase is SE-195, having the mutations as described in Table 6.In embodiments, the polymerase is SE-196, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-197, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-198, having the mutations as described in Table 6. In embodiments,the polymerase is SE-199, having the mutations as described in Table 6.In embodiments, the polymerase is SE-200, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-201, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-202, having the mutations as described in Table 6. In embodiments,the polymerase is SE-203, having the mutations as described in Table 6.In embodiments, the polymerase is SE-204, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-205, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-206, having the mutations as described in Table 6. In embodiments,the polymerase is SE-207, having the mutations as described in Table 6.In embodiments, the polymerase is SE-208, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-209, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-210, having the mutations as described in Table 6. In embodiments,the polymerase is SE-211, having the mutations as described in Table 6.In embodiments, the polymerase is SE-212, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-213, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-214, having the mutations as described in Table 6. In embodiments,the polymerase is SE-215, having the mutations as described in Table 6.In embodiments, the polymerase is SE-216, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-217, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-218, having the mutations as described in Table 6. In embodiments,the polymerase is SE-219, having the mutations as described in Table 6.In embodiments, the polymerase is SE-220, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-221, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-222, having the mutations as described in Table 6. In embodiments,the polymerase is SE-223, having the mutations as described in Table 6.In embodiments, the polymerase is SE-224, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-225, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-226, having the mutations as described in Table 6. In embodiments,the polymerase is SE-227, having the mutations as described in Table 6.In embodiments, the polymerase is SE-228, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-229, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-230, having the mutations as described in Table 6. In embodiments,the polymerase is SE-231, having the mutations as described in Table 6.In embodiments, the polymerase is SE-232, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-233, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-234, having the mutations as described in Table 6. In embodiments,the polymerase is SE-235, having the mutations as described in Table 6.In embodiments, the polymerase is SE-236, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-237, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-238, having the mutations as described in Table 6. In embodiments,the polymerase is SE-239, having the mutations as described in Table 6.In embodiments, the polymerase is SE-240, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-241, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-242, having the mutations as described in Table 6. In embodiments,the polymerase is SE-243, having the mutations as described in Table 6.In embodiments, the polymerase is SE-244, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-245, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-246, having the mutations as described in Table 6. In embodiments,the polymerase is SE-247, having the mutations as described in Table 6.In embodiments, the polymerase is SE-248, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-249, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-250, having the mutations as described in Table 6. In embodiments,the polymerase is SE-251, having the mutations as described in Table 6.In embodiments, the polymerase is SE-252, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-253, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-254, having the mutations as described in Table 6. In embodiments,the polymerase is SE-255, having the mutations as described in Table 6.In embodiments, the polymerase is SE-256, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-257, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-258, having the mutations as described in Table 6. In embodiments,the polymerase is SE-259, having the mutations as described in Table 6.In embodiments, the polymerase is SE-260, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-261, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-262, having the mutations as described in Table 6. In embodiments,the polymerase is SE-263, having the mutations as described in Table 6.In embodiments, the polymerase is SE-264, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-265, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-266, having the mutations as described in Table 6. In embodiments,the polymerase is SE-267, having the mutations as described in Table 6.In embodiments, the polymerase is SE-268, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-269, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-270, having the mutations as described in Table 6. In embodiments,the polymerase is SE-271, having the mutations as described in Table 6.In embodiments, the polymerase is SE-272, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-273, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-274, having the mutations as described in Table 6. In embodiments,the polymerase is SE-275, having the mutations as described in Table 6.In embodiments, the polymerase is SE-276, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-277, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-278, having the mutations as described in Table 6. In embodiments,the polymerase is SE-279, having the mutations as described in Table 6.In embodiments, the polymerase is SE-280, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-281, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-282, having the mutations as described in Table 6. In embodiments,the polymerase is SE-283, having the mutations as described in Table 6.In embodiments, the polymerase is SE-284, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-285, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-286, having the mutations as described in Table 6. In embodiments,the polymerase is SE-287, having the mutations as described in Table 6.In embodiments, the polymerase is SE-288, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-289, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-290, having the mutations as described in Table 6. In embodiments,the polymerase is SE-291, having the mutations as described in Table 6.In embodiments, the polymerase is SE-292, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-293, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-294, having the mutations as described in Table 6. In embodiments,the polymerase is SE-295, having the mutations as described in Table 6.In embodiments, the polymerase is SE-296, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-297, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-298, having the mutations as described in Table 6. In embodiments,the polymerase is SE-299, having the mutations as described in Table 6.In embodiments, the polymerase is SE-300, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-301, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-302, having the mutations as described in Table 6. In embodiments,the polymerase is SE-303, having the mutations as described in Table 6.In embodiments, the polymerase is SE-304, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-305, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-306, having the mutations as described in Table 6. In embodiments,the polymerase is SE-307, having the mutations as described in Table 6.In embodiments, the polymerase is SE-308, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-309, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-310, having the mutations as described in Table 6. In embodiments,the polymerase is SE-311, having the mutations as described in Table 6.In embodiments, the polymerase is SE-312, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-313, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-314, having the mutations as described in Table 6. In embodiments,the polymerase is SE-315, having the mutations as described in Table 6.In embodiments, the polymerase is SE-316, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-317, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-318, having the mutations as described in Table 6. In embodiments,the polymerase is SE-319, having the mutations as described in Table 6.In embodiments, the polymerase is SE-320, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-321, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-322, having the mutations as described in Table 6. In embodiments,the polymerase is SE-323, having the mutations as described in Table 6.In embodiments, the polymerase is SE-324, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-325, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-326, having the mutations as described in Table 6. In embodiments,the polymerase is SE-327, having the mutations as described in Table 6.In embodiments, the polymerase is SE-328, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-329, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-330, having the mutations as described in Table 6. In embodiments,the polymerase is SE-331, having the mutations as described in Table 6.In embodiments, the polymerase is SE-332, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-333, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-334, having the mutations as described in Table 6. In embodiments,the polymerase is SE-335, having the mutations as described in Table 6.In embodiments, the polymerase is SE-336, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-337, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-338, having the mutations as described in Table 6. In embodiments,the polymerase is SE-339, having the mutations as described in Table 6.In embodiments, the polymerase is SE-340, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-341, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-342, having the mutations as described in Table 6. In embodiments,the polymerase is SE-343, having the mutations as described in Table 6.In embodiments, the polymerase is SE-344, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-345, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-346, having the mutations as described in Table 6. In embodiments,the polymerase is SE-347, having the mutations as described in Table 6.In embodiments, the polymerase is SE-348, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-349, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-350, having the mutations as described in Table 6. In embodiments,the polymerase is SE-351, having the mutations as described in Table 6.In embodiments, the polymerase is SE-352, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-353, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-354, having the mutations as described in Table 6. In embodiments,the polymerase is SE-355, having the mutations as described in Table 6.In embodiments, the polymerase is SE-356, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-357, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-358, having the mutations as described in Table 6. In embodiments,the polymerase is SE-359, having the mutations as described in Table 6.In embodiments, the polymerase is SE-360, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-361, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-362, having the mutations as described in Table 6. In embodiments,the polymerase is SE-363, having the mutations as described in Table 6.In embodiments, the polymerase is SE-364, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-365, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-366, having the mutations as described in Table 6. In embodiments,the polymerase is SE-367, having the mutations as described in Table 6.In embodiments, the polymerase is SE-368, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-369, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-370, having the mutations as described in Table 6. In embodiments,the polymerase is SE-371, having the mutations as described in Table 6.In embodiments, the polymerase is SE-372, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-373, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-374, having the mutations as described in Table 6. In embodiments,the polymerase is SE-375, having the mutations as described in Table 6.In embodiments, the polymerase is SE-376, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-377, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-378, having the mutations as described in Table 6. In embodiments,the polymerase is SE-379, having the mutations as described in Table 6.In embodiments, the polymerase is SE-380, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-381, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-382, having the mutations as described in Table 6. In embodiments,the polymerase is SE-383, having the mutations as described in Table 6.In embodiments, the polymerase is SE-384, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-385, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-386, having the mutations as described in Table 6. In embodiments,the polymerase is SE-387, having the mutations as described in Table 6.In embodiments, the polymerase is SE-388, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-389, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-390, having the mutations as described in Table 6. In embodiments,the polymerase is SE-391, having the mutations as described in Table 6.In embodiments, the polymerase is SE-392, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-393, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-394, having the mutations as described in Table 6. In embodiments,the polymerase is SE-395, having the mutations as described in Table 6.In embodiments, the polymerase is SE-396, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-397, havingthe mutations as described in Table 6. In embodiments, the polymerase isSE-398, having the mutations as described in Table 6. In embodiments,the polymerase is SE-399, having the mutations as described in Table 6.In embodiments, the polymerase is SE-400, having the mutations asdescribed in Table 6. In embodiments, the polymerase is SE-401, havingthe mutations as described in Table 6.

In embodiment, the polymerase is SE-28 and includes the following aminoacid substitution mutations relative to SEQ ID NO:1: M129A; D141A;E143A; T144A; L409A; Y410G; P411I; A486V; T515S; T591I; and A640L. Inembodiments, the polymerase is SE-52 and includes the following aminoacid substitution mutations relative to SEQ ID NO:1: M129A; D141A;E143A; L409A; Y410G; A486V; and T515S. In embodiments, the polymerase isSE-53 and includes the following amino acid substitution mutationsrelative to SEQ ID NO:1: M129A; D141A; E143A; L409A; Y410G; A486V; andT591I. In embodiments, the polymerase is SE-56 and includes thefollowing amino acid substitution mutations relative to SEQ ID NO:1:M129A; D141A; E143A; T144A; L409A; Y410G; A486V; and T515S. Inembodiments, the polymerase is SE-58 and includes the following aminoacid substitution mutations relative to SEQ ID NO:1: M129A; D141A;E143A; T144A; L409A; Y410G; A486V; T515S; and T591I. In embodiments, thepolymerase is SE-60 and includes the following amino acid substitutionmutations relative to SEQ ID NO:1: M129A; D141A; E143A; T144A; L409A;Y410G; A486V; T515S; T591; and G153E. In embodiments, the polymerase isSE-61 and includes the following amino acid substitution mutationsrelative to SEQ ID NO:1: M129A; D141A; E143A; T144A; L409A; Y410G;A486V; T515S; T591I; and K713E. In embodiments, the polymerase is SE-62and includes the following amino acid substitution mutations relative toSEQ ID NO:1: M129A; D141A; E143A; T144A; L409A; Y410G; A486V; T515S;T591I; K477W; and K478A. In embodiments, the polymerase is SE-63 andincludes the following amino acid substitution mutations relative to SEQID NO:1: M129A; D141A; E143A; T144A; L409A; Y410G; A486V; T515S; T591I;and A486L. In embodiments, the polymerase is SE-64 and includes thefollowing amino acid substitution mutations relative to SEQ ID NO:1:M129A; D141A; E143A; T144A; L409A; Y410G; A486V; T515S; T591I; andK603A. In embodiments, the polymerase is SE-69 and includes thefollowing amino acid substitution mutations relative to SEQ ID NO:1:M129A; D141A; E143A; T144A; L409A; Y410G; A486V; T515S; T591I; andL479S. In embodiments, the polymerase is SE-69 and includes thefollowing amino acid substitution mutations relative to SEQ ID NO:1:M129A; D141A; E143A; T144A; L409A; Y410G; A486V; T515S; T591I; andA640L.

In embodiments, the polymerase (e.g., a polymerase as described herein)is truncated at the C-terminus. In embodiments, the polymerase (e.g., apolymerase as described herein) is truncated at the C-terminus andretains the ability to incorporate a modified nucleotide. Inembodiments, the polymerase (e.g., a polymerase as described herein) istruncated at the C-terminus, wherein the polymerase is truncated toremove at least 20 amino acids from the C-terminus. In embodiments, thepolymerase (e.g., a polymerase as described herein) is truncated at theC-terminus, wherein the polymerase is truncated to remove at least 10amino acids from the C-terminus. In embodiments, the polymerase (e.g., apolymerase as described herein) is truncated at the C-terminus, whereinthe polymerase is truncated to remove at least 5 amino acids from theC-terminus. In embodiments, the polymerase (e.g., a polymerase asdescribed herein) is truncated at the C-terminus, wherein the truncationremoves 5 to 16 amino acids from the C-terminus. In embodiments, thepolymerase (e.g., a polymerase as described herein) is truncated at theC-terminus, wherein the truncation removes 5 amino acids from theC-terminus. In embodiments, the polymerase (e.g., a polymerase asdescribed herein) is truncated at the C-terminus, wherein the truncationremoves 10 amino acids from the C-terminus. In embodiments, thepolymerase (e.g., a polymerase as described herein) is truncated at theC-terminus, wherein the truncation removes 13 amino acids from theC-terminus. In embodiments, the polymerase (e.g., a polymerase asdescribed herein) is truncated at the C-terminus, wherein the truncationremoves 16 amino acids from the C-terminus.

In another aspect is provided a nucleic acid encoding a mutant orimproved DNA polymerase as described herein, a vector comprising therecombinant nucleic acid, and/or a host cell transformed with thevector. In certain embodiments, the vector is an expression vector. Hostcells comprising such expression vectors are useful in methods of theinvention for producing the mutant or improved polymerase by culturingthe host cells under conditions suitable for expression of therecombinant nucleic acid. The polymerases of the invention may becontained in reaction mixtures and/or kits. The embodiments of therecombinant nucleic acids, host cells, vectors, expression vectors,reaction mixtures and kits are as described above and herein. The fullplasmid nucleic acid sequence used to generate P. horikoshii polymeraseis provided in SEQ ID NO: 2.

In an aspect is provided a kit. Generally, the kit includes at least onecontainer providing a mutant or improved DNA polymerase as describedherein. In embodiments, the kit further includes one or more additionalcontainers providing one or more additional reagents. For example, theone or more additional containers provide nucleoside triphosphates; abuffer suitable for polynucleotide extension; and/or a primerhybridizable, under polynucleotide extension conditions, to apredetermined polynucleotide template. The kit may also include atemplate nucleic acid (DNA and/or RNA), one or more primer or probepolynucleotides, nucleoside triphosphates (including, e.g.,deoxyribonucleotides, ribonucleotides, labelled nucleotides, modifiednucleotides), buffers, salts, labels (e.g., fluorophores). Inembodiments, the kit includes a nucleotide solution as described herein.In embodiments, the kit further includes instructions.

In an aspect is provided a nucleotide sequence useful for identifyingpolymerase mutants capable of rapid nucleotide incorporation. Inembodiments, the sequences are described in Table 2.

In an aspect is provided a nucleic acid sequence encoding the polymeraseenzymes as described herein. In embodiments, the nucleic acid sequenceis SEQ ID NO:3, wherein the nucleic acid sequence includesconservatively modified variants to encode to appropriate amino acidmutation as described herein. For example, given that the wild typenucleotide sequence encoding Pyrococcus horikoshii (SEQ ID NO:2)polymerase is known, it is possible to deduce a nucleotide sequenceencoding any given mutant version of Pyrococcus horikoshii having one ormore amino acid substitutions using the standard genetic code.Similarly, nucleotide sequences can readily be derived for mutantversions other polymerases such as those species described in Table 7.Nucleic acid molecules having the required nucleotide sequence may thenbe constructed using standard molecular biology techniques known in theart. The nucleic acid sequence described herein may also,advantageously, be included in a suitable expression vector to expressthe polymerase proteins encoded therefrom in a suitable host.Incorporation of cloned DNA into a suitable expression vector forsubsequent transformation of said cell and subsequent selection of thetransformed cells is well known to those skilled in the art as providedin Sambrook et al. (1989), Molecular cloning: A Laboratory Manual, ColdSpring Harbour Laboratory. In embodiments, the nucleic acid sequence isSEQ ID NO:2, wherein the nucleic acid sequence includes conservativelymodified variants to encode to appropriate amino acid mutation asdescribed herein. Such an expression vector includes a vector having anucleic acid according to the invention operably linked to regulatorysequences, such as promoter regions, that are capable of effectingexpression of said DNA fragments. The nucleic acid sequence may encode amature protein or a protein having a prosequence, including thatencoding a leader sequence on the preprotein which is then cleaved bythe host cell to form a mature protein. The vectors may be plasmid,virus or phage vectors provided with an origin of replication, andoptionally a promoter for the expression of said nucleotide andoptionally a regulator of the promoter. The vectors may contain one ormore selectable markers (e.g., an antibiotic resistance gene).

METHODS OF USE

In an aspect, a method of incorporating a modified nucleotide into anucleic acid sequence is provided. The method includes allowing thefollowing components to interact: (i) a nucleic acid template, (ii) aprimer that has an extendible 3′ end, (iii) a nucleotide solution, and(iv) a polymerase (e.g., a DNA polymerase or a thermophilic nucleic acidpolymerase as described here). The polymerase used in the methodincludes an amino acid sequence that is at least 80% identical to acontinuous 500 amino acid sequence within SEQ ID NO: 1. In embodiments,the polymerase has exonuclease activity that is reduced at least 80%relative to the exonuclease activity of a polymerase of SEQ ID NO: 1. Inembodiments, the polymerase includes an amino acid sequence that is atleast 80% identical to a continuous 500 amino acid sequence within SEQID NO: 1. The polymerase includes substitution mutations at positions141 and 143 of SEQ ID NO: 1. The polymerase further includes at leastone amino acid substitution mutation at a position selected frompositions 409, 410, and 411 of SEQ ID NO: 1. In embodiments, thepolymerase includes an amino acid sequence that is at least 80%identical to a continuous 500 amino acid sequence within SEQ ID NO: 1.The polymerase includes substitution mutations at positions 129, 141,143, and 486 of SEQ ID NO: 1. The polymerase further includes at leastone amino acid substitution mutation at a position selected frompositions 409, 410, and 411 of SEQ ID NO: 1. In embodiments, thepolymerase includes an amino acid sequence that is at least 80%identical to a continuous 500 amino acid sequence within SEQ ID NO: 1.The polymerase includes substitution mutations at positions 141, 143,and 153 of SEQ ID NO: 1. The polymerase further includes at least oneamino acid substitution mutation at a position selected from positions409, 410, and 411 of SEQ ID NO: 1. In embodiments, the polymeraseincludes an amino acid sequence that is at least 80% identical to acontinuous 500 amino acid sequence within SEQ ID NO: 1. The polymeraseincludes substitution mutations at positions 129, 141, 143, 153, and 486of SEQ ID NO: 1. The polymerase further includes at least one amino acidsubstitution mutation at a position selected from positions 409, 410,and 411 of SEQ ID NO: 1. In embodiments, the polymerase is a polymeraseas described herein (e.g., in an embodiment, claim, or SEQ ID).

In an aspect, a method of preparing a growing polynucleotidecomplementary to a target single-stranded polynucleotide in a sequencingreaction is provided. The method incudes incorporating a modifiednucleotide molecule into a growing complementary polynucleotide, wherethe incorporation of the modified nucleotide prevents the introductionof any subsequent nucleotide into the growing complementarypolynucleotide and wherein the incorporation of the modified nucleotidemolecule is accomplished by a polymerase as described herein.

In an aspect, a method for performing a primer extension reaction isprovided. The method incudes contacting a modified polymerase comprisingthe amino acid sequence of any one of the polymerases described hereinor in Table 7, with a nucleic acid molecule and a modified nucleotideunder conditions where the modified nucleotide is incorporated into thenucleic acid molecule by the polymerase.

In an aspect, provided herein is a method of sequencing a nucleic acidsequence including a) hybridizing a nucleic acid template with a primerto form a primer-template hybridization complex; b) contacting theprimer-template hybridization complex with a DNA polymerase andnucleotides, wherein the DNA polymerase is a polymerase according to anyof the various embodiments described herein and the nucleotides comprisea modified nucleotide, wherein the modified nucleotide comprises adetectable label; c) subjecting the primer-template hybridizationcomplex to conditions which enable the polymerase to incorporate amodified nucleotide into the primer-template hybridization complex toform a modified primer-template hybridization complex; and d) detectingthe detectable label; thereby sequencing a nucleic acid

In embodiments, the nucleic acid template is DNA, RNA, or analogsthereof. In embodiments, the nucleic acid template includes a primerhybridized to the template. In embodiments, the nucleic acid template isa primer. Primers are usually single-stranded for maximum efficiency inamplification, but may alternatively be double-stranded. Ifdouble-stranded, the primer is usually first treated to separate itsstrands before being used to prepare extension products. Thisdenaturation step is typically affected by heat, but may alternativelybe carried out using alkali, followed by neutralization. Thus, a“primer” is complementary to a nucleic acid template, and complexes byhydrogen bonding or hybridization with the template to give aprimer/template complex for initiation of synthesis by a polymerase,which is extended by the addition of covalently bonded bases linked atits 3′ end complementary to the template in the process of DNAsynthesis. The DNA template for a sequencing reaction will typicallycomprise a double-stranded region having a free 3′ hydroxyl group whichserves as a primer or initiation point for the addition of furthernucleotides in the sequencing reaction. The region of the DNA templateto be sequenced will overhang this free 3′ hydroxyl group on thecomplementary strand. The primer bearing the free 3′ hydroxyl group maybe added as a separate component (e.g. a short oligonucleotide) whichhybridises to a region of the template to be sequenced. Alternatively,the primer and the template strand to be sequenced may each form part ofa partially self-complementary nucleic acid strand capable of forming anintramolecular duplex, such as for example a hairpin loop structure.Nucleotides are added successively to the free 3′ hydroxyl group,resulting in synthesis of a polynucleotide chain in the 5′ to 3′direction. After each nucleotide addition the nature of the base whichhas been added will be determined, thus providing sequence informationfor the DNA template.

In embodiments, the nucleotide solution includes modified nucleotides.It is understood that a modified nucleotide and a nucleotide analogueare interchangeable terminology in this context. In embodiments, thenucleotide solution includes labelled nucleotides. In embodiments, thenucleotides include synthetic nucleotides. In embodiments, thenucleotide solution includes modified nucleotides that independentlyhave different reversible terminating moieties (e.g., nucleotide A hasan A-term reversible terminator, nucleotide G has an S-term reversibleterminator, nucleotide C has an S-term reversible terminator, andnucleotide T has an i-term1 reversible terminator). In embodiments thenucleotide solution contains native nucleotides. In embodiments thenucleotide solution contains labelled nucleotides.

In embodiments, the modified nucleotide has a removable group, forexample a label, a blocking group, or protecting group. The removablegroup includes a chemical group that can be removed from a dNTP analoguesuch that a DNA polymerase can extend the nucleic acid (e.g., a primeror extension product) by the incorporation of at least one additionalnucleotide. In embodiments, the removal group is a reversibleterminator.

In embodiments, the modified nucleotide includes a blocking moietyand/or a label moiety. The blocking moiety on a nucleotide can bereversible, whereby the blocking moiety can be removed or modified toallow the 3′ hydroxyl to form a covalent bond with the 5′ phosphate ofanother nucleotide. The blocking moiety can be effectively irreversibleunder particular conditions used in a method set forth herein. A labelmoiety of a nucleotide can be any moiety that allows the nucleotide tobe detected, for example, using a spectroscopic method. In embodiments,one or more of the above moieties can be absent from a nucleotide usedin the methods and compositions set forth herein. For example, anucleotide can lack a label moiety or a blocking moiety or both.

In embodiments, the blocking moiety can be located, for example, at the3′ position of the nucleotide and may be a chemically cleavable moietysuch as an allyl group, an azidomethyl group or a methoxymethyl group,or may be an enzymatically cleavable group such as a phosphate. Suitablenucleotide blocking moieties are described in applications WO2004/018497, U.S. Pat. Nos. 7,057,026, 7,541,444, WO 96/07669, U.S. Pat.Nos. 5,763,594, 5,808,045, 5,872,244 and 6,232,465, the contents ofwhich are incorporated herein by reference in their entirety. Thenucleotides may be labelled or unlabeled. In embodiments, the modifiednucleotides with reversible terminators useful in methods providedherein may be 3′-O-blocked reversible or 3′-unblocked reversibleterminators. The 3′-O-blocked reversible terminators are known in theart, and may be, for instance, a 3′-ONH₂ reversible terminator, a3′-O-allyl reversible terminator, or a 3′-O-azidomethyl reversibleterminator.

In embodiments, the modified nucleotides useful in methods providedherein can include 3′-unblocked reversible terminators. The 3′-unblockedreversible terminators are known in the art and include for example, the“virtual terminator” as described in U.S. Pat. No. 8,114,973 and the“lightening terminator” as described in U.S. Pat. No. 10,041,115, thecontents of which are incorporated herein by reference in theirentirety.

In embodiments, the modified nucleotide (also referred to herein as anucleotide analogue) has the formula

wherein Base is a Base as described herein (e.g., B of Formula Ia orIb), R³ is —OH, monophosphate, or polyphosphate or a nucleic acid, andR′ is a reversible terminator having the formula:

wherein R^(A) and R^(B) are hydrogen or alkyl and R^(C) is the remainderof the reversible terminator. In embodiments, the reversible terminatoris

In embodiments, the reversible terminator is

In embodiments, the reversible terminator is

In embodiments, the reversible terminator is

In embodiments, the reversible terminator is

In embodiments, the reversible terminator is

In embodiments, the reversible terminator is

In embodiments, the reversible terminator is

In embodiments, the reversible terminator is

In embodiments, the reversible terminator is

In embodiments, the modified nucleotide (e.g., also referred to hereinas a nucleotide analogue) has the formula:

The symbol “----” is a non-covalent bond. The symbol B is a base oranalogue thereof. L² is a covalent linker (e.g., a cleavable linker). L³is a covalent linker. L⁴ is a bond, substituted or unsubstitutedalkylene, substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene. R² is hydrogen or —OR^(2A),wherein R^(2A) is hydrogen, polymerase-compatible moiety, orpolymerase-compatible cleavable moiety. R³ is —OH, monophosphate, orpolyphosphate or a nucleic acid. R⁴ is substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl. R⁵ is a detectable label, an anchor moiety, or affinityanchor moiety. R¹² is a complementary affinity anchor moiety binder. R¹³is a detectable label. The symbols X¹ and X² are independently hydrogen,halogen, —N₃, or —CN, wherein at least wherein at least one of X¹ or X²is halogen, —N₃, or —CN. In embodiments, at least one of X¹ or X² ishalogen. In embodiments, if X¹ is —N₃ then X² is not —N₃ for formula(Ia), (Ib), and (Ic).

In embodiments, the nucleotide analogue has the formula:

wherein R³, B, R², X¹, X², L², R⁵, R¹², L³, and R¹³ are as describedherein, including embodiments.

In embodiments, the nucleotide analogue has the formula:

wherein R³, B, R², X₁, X², L², L⁴, R⁴, R⁵, R¹², L³, and R¹³ are asdescribed herein, including embodiments.

In embodiments, the nucleotide analogue has the formula:

wherein R³, B, R², X¹, X², L², L⁴, R⁴, R⁴, R¹², L³, and R¹³ are asdescribed herein, including embodiments.

In embodiments, the nucleotide analogue has the formula:

wherein R³, B, R², X¹, and X² are as described herein, includingembodiments. In embodiments, the nucleotide analogue has the formula:

wherein R³, B, L², R⁵, R², X¹, and X² are as described herein, includingembodiments. In embodiments, the nucleotide analogue has the formula:

wherein R³, B, L², R⁵, R¹², L³, R¹³, R², X¹, and X² are as describedherein, including embodiments. In embodiments, the nucleotide analoguehas the formula:

wherein R³, B, R², X¹, X², L4, and R4 are as described herein, includingembodiments. In embodiments, the nucleotide analogue has the formula:

wherein R³, B, L², R⁵, R², X¹, X², L⁴, and R⁴ are as described herein,including embodiments. In embodiments, the nucleotide analogue has theformula:

wherein R³, B, L², R⁵, R¹², L³, R¹³, R², X¹, X², L⁴ and R⁴ are asdescribed herein, including embodiments. In embodiments, the nucleotideanalogue has the formula:

wherein R³, B, R², X¹, X², L⁴ and R⁵ are as described herein, includingembodiments. In embodiments, the nucleotide analogue has the formula:

wherein R³, B, R², X¹, X², L⁴, R⁵, R¹², L³, and R¹³ are as describedherein, including embodiments.

In embodiments, B is,

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, B¹ is

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, R³ is a monophosphate moiety. In embodiments, R³ is atriphosphate moiety.

In embodiments, X¹ is hydrogen. In embodiments, X¹ is halogen (e.g.,—F). In embodiments, X¹ is —CN. In embodiments, X¹ is —N₃. Inembodiments, X² is hydrogen. In embodiments, X² is halogen (e.g., —F).In embodiments, X² is —CN. In embodiments, X² is —N₃.

In embodiments, X¹ is hydrogen, and X² is halogen. In embodiments, X¹ ishydrogen, and X² is —CN. In embodiments, X¹ is hydrogen, and X² is —N₃.In embodiments, X¹ is halogen, and X² is hydrogen. In embodiments, X¹ ishalogen, and X² is halogen. In embodiments, X¹ is halogen, and X² is—CN. In embodiments, X¹ is halogen, and X² is —N₃. In embodiments, X¹ is—CN, and X² is hydrogen. In embodiments, X¹ is —CN, and X² is halogen.In embodiments, X¹ is —CN, and X² is —CN. In embodiments, X¹ is —CN, andX² is —N₃. In embodiments, X¹ is —N₃, and X² is hydrogen. Inembodiments, X¹ is —N₃, and X² is halogen. In embodiments, X¹ is —N₃,and X² is —CN. In embodiments, X¹ is —N₃, and X² is —N₃.

In embodiments, X¹ is hydrogen, and X² is —F. In embodiments, X¹ ishydrogen, and X² is —CN. In embodiments, X¹ is hydrogen, and X² is —N₃.In embodiments, X¹ is —F, and X² is hydrogen. In embodiments, X¹ is —F,and X² is —F. In embodiments, X¹ is —F, and X² is —CN. In embodiments,X¹ is —F, and X² is —N₃. In embodiments, X¹ is —CN, and X² is hydrogen.In embodiments, X¹ is —CN, and X² is —F. In embodiments, X¹ is —CN, andX² is —CN. In embodiments, X¹ is —CN, and X² is —N₃. In embodiments, X¹is —N₃, and X² is hydrogen. In embodiments, X¹ is —N₃, and X² is —F. Inembodiments, X¹ is —N₃, and X² is —CN. In embodiments, X¹ is —N₃, and X²is —N₃.

In embodiments, X¹ is H and X² is —N₃. In embodiments, X¹ is H and X² is—CN. In embodiments, X¹ is H and X² is —F. In embodiments, X¹ is —F andX² is —F. In embodiments, X¹ is —N₃ and X² is —N₃. In embodiments, X¹ is—N₃ and X² is —N₃. In embodiments, X¹ is —N₃ and X² is —CN. Inembodiments, X¹ is —CN and X² is —CN.

In embodiments, X¹ is H and X² is —N₃ for formula (Ia), (Ib), and (Ic).In embodiments, X¹ is H and X² is —CN for formula (Ia), (Ib), and (Ic).In embodiments, X¹ is H and X² is —F for formula (Ia), (Ib), and (Ic).In embodiments, X¹ is —F and X² is —F for formula (Ia), (Ib), and (Ic).In embodiments, X¹ is —N₃ and X² is —N₃ for formula (Ia), (Ib), and(Ic). In embodiments, X¹ is —N₃ and X² is —N₃ for formula (Ia), (Ib),and (Ic). In embodiments, X¹ is —N₃ and X² is —CN for formula (Ia),(Ib), and (Ic). In embodiments, X¹ is —CN and X² is —CN for formula(Ia), (Ib), and (Ic).

In embodiments, X¹ is not —N₃ and X² is not —N₃ for formula (Ia), (Ib),and (Ic). In embodiments, X¹ is not —CN and X² is not —N₃ for formula(Ia), (Ib), and (Ic). In embodiments, X¹ is not —CN and X² is not —CNfor formula (Ia), (Ib), and (Ic).

In embodiments, X¹ is not —N₃ and X² is not —N₃ for formula (IIa),(IIb), (IIc), (IIIa), or (IIIb). In embodiments, X¹ is not —N₃ and X² isnot —CN for formula (IIa), (IIb), (IIc), (IIIa), or (IIIb). Inembodiments, X¹ is not —CN and X² is not —CN for formula (IIa), (IIb),(IIc), (IIIa), or (IIIb).

In embodiments, L² is a cleavable linker. In embodiments, L² is anon-cleavable linker. In embodiments, L² is a chemically cleavablelinker. In embodiments, L² is a photocleavable linker, an acid-cleavablelinker, a base-cleavable linker, an oxidant-cleavable linker, areductant-cleavable linker, or a fluoride-cleavable linker. Inembodiments, L² is a cleavable linker comprising a dialkylketal linker,an azo linker, an allyl linker, a cyanoethyl linker, a1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl linker, or anitrobenzyl linker.

In embodiments, L² is a bond, substituted or unsubstituted alkylene,substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene.

In embodiments, L² is

In embodiments, L² is

In embodiments, L² is

In embodiments, L² is

In embodiments, L² is

In embodiments, L² is

In embodiments, L² is

In embodiments, L² is

In embodiments, L² is

In embodiments, L² is

In embodiments, L² is

In embodiments, L² is

In embodiments, L² is

In embodiments, L² is

In embodiments, L² is

In embodiments, L² is

In embodiments, L² is

In embodiments, L² is

In embodiments, L² is

In embodiments, L² is

H In embodiments, L² is

In embodiments, L² is

In embodiments L² is

In embodiments, L² is

In embodiments, L² is

In embodiments, L² is

In embodiments, L² is

In embodiments, L² is

In embodiments, L² is

In embodiments, L² is

In embodiments, L² is

In embodiments, L² is

In embodiments, -L²-R⁵ is

In embodiments -L²-R⁵ is

In embodiments, -L²-R⁵ is

In embodiments, -L²-R⁵ is

In embodiments, -L²-R⁵ is

In embodiments, R⁵ is a streptavidin moiety. In embodiments, R⁵ is ananchor moiety, or affinity anchor moiety. In embodiments, R⁵ is ananchor moiety. In embodiments, R⁵ is an affinity anchor moiety.

In embodiments, R⁵ is

unsubstituted ethynyl,

In embodiments, R⁵ is

In embodiments, R⁵ is a detectable label. In embodiments, R⁵ is afluorescent dye. In embodiments, R⁵ is an anchor moiety. In embodiments,R⁵ is a click chemistry reactant moiety. In embodiments, R⁵ is atrans-cyclooctene moiety or azide moiety. In embodiments, R⁵ is anaffinity anchor moiety. In embodiments, R⁵ is a biotin moiety. Inembodiments, R⁵ is a reactant for a bioconjugate reaction that forms acovalent bond between R⁵ and a second bioconjugate reaction reactant(e.g., R¹²).

In embodiments, R⁵ is a fluorescent dye. In embodiments R⁵ is a AlexaFluor® 350 moiety, Alexa Fluor® 405 moiety, Alexa Fluor® 430 moiety,Alexa Fluor® 488 moiety, Alexa Fluor® 532 moiety, Alexa Fluor® 546moiety, Alexa Fluor® 555 moiety, Alexa Fluor® 568 moiety, Alexa Fluor®594 moiety, Alexa Fluor® 610 moiety, Alexa Fluor® 633 moiety, AlexaFluor® 635 moiety, Alexa Fluor® 647 moiety, Alexa Fluor® 660 moiety,Alexa Fluor® 680 moiety, Alexa Fluor® 700 moiety, Alexa Fluor® 750moiety, or Alexa Fluor® 790 moiety. In embodiments the detectable moietyis a Alexa Fluor® 488 moiety, Rhodamine 6G (R6G) moiety, ROX ReferenceDye (ROX) moiety, or Cy5 moiety.

In embodiments R⁵ is a FAM™ moiety, TET™ moiety, JOE™ moiety, VIC®moiety, HEX™ moiety, NED™ moiety, PET® moiety, ROX™ moiety, TAMRA™moiety, TET™ moiety, Texas Red® moiety, Alexa Fluor® 488 moiety,Rhodamine 6G (R6G) moiety, ROX Reference Dye (ROX) moiety, Sulfo-Cy5, orCy5 moiety. In embodiments R⁵ is a Rhodamine 6G (R6G) moiety, ROXReference Dye (ROX) moiety, Sulfo-Cy5, or Cy5 moiety.

In embodiments, R⁵ is a biotin moiety. In embodiments, R⁵ is a biotinmoiety and R¹² is a streptavidin moiety.

In embodiments, R⁵ is

In embodiments, R⁵ is

In embodiments, L⁴ is a bond, substituted or unsubstituted alkylene,substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene. In embodiments, L⁴ is abond, substituted or unsubstituted C₁-C₈ alkylene, substituted orunsubstituted 2 to 8 membered heteroalkylene, substituted orunsubstituted C₃-C₈ cycloalkylene, substituted or unsubstituted 3 to 8membered heterocycloalkylene, substituted or unsubstituted C₆-C₁₀arylene, or substituted or unsubstituted 5 to 10 membered heteroarylene.In embodiments, L⁴ is a substituted or unsubstituted C₁-C₆ alkylene orsubstituted or unsubstituted 2 to 6 membered heteroalkylene. Inembodiments, L⁴ is an unsubstituted C₁-C₄ alkylene. In embodiments, L⁴is a bond.

In embodiments, L⁴ is a substituted or unsubstituted methylene. Inembodiments, L⁴ is substituted with a substituted or unsubstituted C₁-C₆alkylene, substituted or unsubstituted 2 to 6 membered heteroalkylene,substituted or unsubstituted C₃-C₆ cycloalkylene, substituted orunsubstituted 3 to 6 membered heterocycloalkylene, substituted orunsubstituted phenyl, or substituted or unsubstituted 5 to 6 memberedheteroarylene. In embodiments, L⁴ is a bond, substituted orunsubstituted C₁-C₆ alkylene, substituted or unsubstituted 2 to 6membered heteroalkylene, substituted or unsubstituted C₃-C₆cycloalkylene, substituted or unsubstituted 3 to 6 memberedheterocycloalkylene, substituted or unsubstituted phenyl, or substitutedor unsubstituted 5 to 6 membered heteroarylene.

In embodiments, L⁴ is substituted with a substituted or unsubstitutedC₁-C₆ alkylene or substituted or unsubstituted 2 to 6 memberedheteroalkylene. In embodiments, L⁴ is a substituted or unsubstitutedC₁-C₆ alkylene or substituted or unsubstituted 2 to 6 memberedheteroalkylene. In embodiments, L⁴ is a substituted or unsubstitutedmethylene. In embodiments, L⁴ is substituted with a substituted orunsubstituted C₁-C₆ alkylene. In embodiments, L⁴ is an unsubstitutedmethylene.

In embodiments, R⁴ is substituted or unsubstituted C₁-C₆ alkyl,substituted or unsubstituted 2 to 6 membered heteroalkyl, substituted orunsubstituted C₃-C₆ cycloalkyl, substituted or unsubstituted 3 to 6membered heterocycloalkyl, substituted or unsubstituted phenyl, orsubstituted or unsubstituted 5 to 6 membered heteroaryl. In embodiments,R⁴ is substituted or unsubstituted C₁-C₆ alkyl, or substituted orunsubstituted phenyl. In embodiments, R⁴ is an unsubstituted C₁-C₆alkyl.

In embodiments, R⁴ is substituted (e.g., substituted with a substituentgroup, a size-limited substituent group, or lower substituent group) orunsubstituted alkyl. In embodiments, R⁴ is substituted (e.g.,substituted with a substituent group, a size-limited substituent group,or lower substituent group) alkyl. In embodiments, R⁴ is unsubstitutedalkyl. In embodiments, R⁴ is substituted or unsubstituted alkyl (e.g.,C₁-C₈, C₁-C₆, C₁-C₄, or C₁-C₂). In embodiments, R⁴ is substituted alkyl(e.g., C₁-C₈, C₁-C₆, C₁-C₄, or C₁-C₂). In embodiments, R⁴ isunsubstituted alkyl (e.g., C₁-C₈, C₁-C₆, C₁-C₄, or C₁-C₂).

In embodiments, R⁴ is substituted (e.g., substituted with a substituentgroup, a size-limited substituent group, or lower substituent group) orunsubstituted heteroalkyl. In embodiments, R⁴ is substituted (e.g.,substituted with a substituent group, a size-limited substituent group,or lower substituent group) heteroalkyl. In embodiments, R⁴ isunsubstituted heteroalkyl. In embodiments, R⁴ is substituted orunsubstituted heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to6 membered, 2 to 3 membered, or 4 to 5 membered). In embodiments, R⁴ issubstituted heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6membered, 2 to 3 membered, or 4 to 5 membered). In embodiments, R⁴ is anunsubstituted heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to6 membered, 2 to 3 membered, or 4 to 5 membered).

In embodiments, R⁴ is substituted or unsubstituted alkyl (e.g., C₁-C₈alkyl, C₁-C₆ alkyl, or C₁-C₄ alkyl). In embodiments, R⁴ is substitutedalkyl (e.g., C₁-C₈ alkyl, C₁-C₆ alkyl, or C₁-C₄ alkyl). In embodiments,R⁴ is an unsubstituted alkyl (e.g., C₁-C₈ alkyl, C₁-C₆ alkyl, or C₁-C₄alkyl). In embodiments, R⁴ is substituted or unsubstituted methyl. Inembodiments, R⁴ is substituted or unsubstituted C₂ alkyl. Inembodiments, R⁴ is substituted or unsubstituted C₃ alkyl. Inembodiments, R⁴ is substituted or unsubstituted C₄ alkyl. Inembodiments, R⁴ is substituted or unsubstituted C₅ alkyl. Inembodiments, R⁴ is substituted or unsubstituted C₆ alkyl. Inembodiments, R⁴ is substituted or unsubstituted C₇ alkyl. Inembodiments, R⁴ is substituted or unsubstituted C₈ alkyl. Inembodiments, R⁴ is substituted methyl. In embodiments, R⁴ is substitutedC₂ alkyl. In embodiments, R⁴ is substituted C₃ alkyl. In embodiments, R⁴is substituted C₄ alkyl. In embodiments, R⁴ is substituted C₅ alkyl. Inembodiments, R⁴ is substituted C₆ alkyl. In embodiments, R⁴ issubstituted C₇ alkyl. In embodiments, R⁴ is substituted C₈ alkyl. Inembodiments, R⁴ is an unsubstituted methyl. In embodiments, R⁴ is anunsubstituted C₂ alkyl. In embodiments, R⁴ is an unsubstituted C₃ alkyl.In embodiments, R⁴ is an unsubstituted C₄ alkyl (e.g., t-butyl). Inembodiments, R⁴ is an unsubstituted C₅ alkyl. In embodiments, R⁴ is anunsubstituted C₆ alkyl. In embodiments, R⁴ is an unsubstituted C₇ alkyl.In embodiments, R⁴ is an unsubstituted C₈ alkyl.

In embodiments, the nucleotide analogue has the formula:

wherein B and X¹ are as described herein, including embodiments. Inembodiments, the nucleotide analogue has the formula:

wherein B and R^(C) are as described herein, including embodiments. Inembodiments, the nucleotide analogue has the formula:

wherein B and R^(C) are as described herein, including embodiments. Inembodiments, the nucleotide analogue has the formula:

wherein R², R³, B, L², L⁵, and R^(C) are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein X¹ is as described herein, including embodiments.

In embodiments, the nucleotide analogue has the formula:

wherein B and X¹ are as described herein, including embodiments.

In embodiments, the nucleotide analogue has the formula:

wherein X¹ is as described herein, including embodiments.

In embodiments, the nucleotide analogue has the formula:

wherein B and X¹ are as described herein, including embodiments.

In embodiments, the nucleotide analogue has the formula:

wherein X¹ is as described herein, including embodiments.

In embodiments, the nucleotide analogue has the formula:

wherein B and X² are as described herein, including embodiments.

In embodiments, the nucleotide analogue has the formula:

wherein X² is as described herein, including embodiments.

In embodiments, the nucleotide analogue has the formula:

wherein X¹ is as described herein, including embodiments.

In embodiments, the nucleotide analogue has the formula:

wherein B and X¹ are as described herein, including embodiments.

In embodiments, the nucleotide analogue has the formula:

wherein X¹ is as described herein, including embodiments.

In embodiments, the nucleotide analogue has the formula:

wherein X¹, B, L⁴, and R⁴ are as described herein, includingembodiments.

In embodiments, the nucleotide analogue has the formula:

wherein X¹, B, L⁴, and R⁴ are as described herein, includingembodiments.

In embodiments, the nucleotide analogue has the formula:

wherein X¹, B, L⁴, and R⁴ are as described herein, includingembodiments.

In embodiments, the nucleotide analogue has the formula:

wherein X², B, L⁴, and R⁴ are as described herein, includingembodiments.

In embodiments, the nucleotide analogue has the formula:

wherein X¹, L⁴, and R⁴ are as described herein, including embodiments.

In embodiments, the nucleotide analogue has the formula:

wherein X¹, B, L⁴, and R⁵ are as described herein, includingembodiments.

In embodiments, the nucleotide analogue has the formula:

wherein X², B, L⁴, and R⁵ are as described herein, includingembodiments.

In embodiments, the nucleotide analogue has the formula:

wherein X¹, B, L⁴, and R⁵ are as described herein, includingembodiments.

In embodiments, the nucleotide analogue has the formula:

wherein X¹, L⁴, and R⁵ are as described herein, including embodiments.

In embodiments, the nucleotide analogue has the formula:

wherein X and X¹ are each independently a halogen, and L⁴, and R⁴ are asdescribed herein.

In embodiments, the polymerases provided herein are used with modifiednucleotides and nucleic acid synthesis methods as described in US2018/0274024, WO/2017/058953, WO 2017/205336, or US 2019/0077726, thecontents of which are incorporated by reference herein for all purposes.

In embodiments, methods of incorporating a modified nucleotide into anucleic acid sequence provided herein includes a polymerase (a syntheticor variant DNA polymerase) according to any of the various embodimentsdescribed herein.

In embodiments, mutations may include substitution of the amino acid inthe parent amino acid sequences with an amino acid, which is not theparent amino acid. In embodiments, the mutations may result inconservative amino acid changes. In embodiments, non-polar amino acidsmay be converted into polar amino acids (threonine, asparagine,glutamine, cysteine, tyrosine, aspartic acid, glutamic acid orhistidine) or the parent amino acid may be changed to an alanine.

In embodiments, the method includes maintaining the temperature at about55° C. In embodiments, the method includes maintaining the temperatureat about 55° C. to about 80° C. In embodiments, the method includesmaintaining the temperature at about 60° C. to about 70° C. Inembodiments, the method includes maintaining the temperature at about65° C. to about 75° C. In embodiments, the method includes maintainingthe temperature at about 65° C. In embodiments, the method includesmaintaining the temperature at a pH of 8.0 to 11.0. In embodiments, thepH is 9.0 to 11.0. In embodiments, the pH is 9.5. In embodiments, the pHis 10.0. In embodiments, the pH is 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, or11.0. In embodiments, the pH is from 9.0 to 11.0, and the temperature isabout 60° C. to about 70° C.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

EXAMPLES Example 1: Development of Family B DNA Polymerase Variants

Despite ongoing research, current modifications to the DNA polymerasestill do not show sufficiently high incorporation rates of modifiednucleotides. In nucleic acid sequencing applications, the modifiednucleotide typically has a reversible terminator at the 3′ position anda modified base (e.g., a base linked to a fluorophore via a cleavablelinker). In case of cleavable linkers attached to the base, there isusually a residual spacer arm left after the cleavage. This residualmodification may interfere with incorporation of subsequent nucleotidesby polymerase. Therefore, it is highly desirable to have polymerases forcarrying out sequencing by synthesis process (SBS) that are tolerable ofthese scars. In addition to rapid incorporation, the enzyme needs to bestable and have high incorporation fidelity. Balancing incorporationkinetics and fidelity is a challenge. If the mutations in the polymeraseresult in a rapid average incorporation half-time but is too promiscuoussuch that the inappropriate nucleotide is incorporated into the primer,this will result in a large source of error in sequencing applications.It is also desirable to design a polymerase capable to incorporating avariety of reversible terminators. Discovering a polymerase that hassuitable kinetics and low misincorporation error remains a challenge.

An aim of the general experimental plan was to produce a robust,optimized polymerase for nucleic acid sequencing methods. DNApolymerases of the Pyrococcus genus share similar anerobic features asother thermophilic genera (e.g., Archaeoglobus, Thermoautotrophican,Methanococcus) however Pyrococcus species thrive in higher temperatures,ca 100° C., and tolerate extreme pressures. For example, the area aroundundersea hot vents, where P. abyssi has been found, there is nosunlight, the temperature is around 98° C.-100° C. and the pressure isabout 200 atm. These Pyrococcus polymerases possess inherent propertiesthat are beneficial for sequencing applications.

In the context of nucleic acid sequencing, the use of nucleotidesbearing a 3′ reversible terminator allows successive nucleotides to beincorporated into a polynucleotide chain in a controlled manner. The DNAtemplate for a sequencing reaction will typically comprise adouble-stranded region having a free 3′ hydroxyl group which serves as aprimer or initiation point for the addition of further nucleotides inthe sequencing reaction. The region of the DNA template to be sequencedwill overhang this free 3′ hydroxyl group on the complementary strand.The primer bearing the free 3′ hydroxyl group may be added as a separatecomponent (e.g. a short oligonucleotide) which hybridizes to a region ofthe template to be sequenced. Following the addition of a singlenucleotide to the DNA template, the presence of the 3′ reversibleterminator prevents incorporation of a further nucleotide into thepolynucleotide chain. While the addition of subsequent nucleotides isprevented, the identity of the incorporated is detected (e.g., excitinga unique detectable label that is linked to the incorporatednucleotide). The reversible terminator is then removed, leaving a free3′ hydroxyl group for addition of the next nucleotide. The sequencingcycle can then continue with the incorporation of the next blocked,labelled nucleotide.

Sequencing by synthesis of nucleic acids ideally requires the controlled(i.e. one at a time), yet rapid, incorporation of the correctcomplementary nucleotide opposite the oligonucleotide being sequenced.This allows for accurate sequencing by adding nucleotides in multiplecycles as each nucleotide residue is sequenced one at a time, thuspreventing an uncontrolled series of incorporations occurring.

As described herein wild-type Pyrococcus enzymes (e.g., P. horikoshiiand P. abyssi) have difficulty incorporating modified nucleotides (e.g.,nucleotides including a reversible terminator and/or a cleavable linkedbase). A minimum set of mutations is necessary to modify the exonucleaseactivity and permit incorporation (e.g., the mutations found in SE-1).The modifications at amino acid positions D141 and E143 (relative towild-type) are known to affect exonuclease activity (designated exo-)(see, for example, U.S. Pat. No. 5,756,334 and Southworth et al, 1996Proc. Natl Acad. Sci USA 93:5281). This 3′-5′ exonuclease activity isabsent in some DNA polymerases (e.g., Taq DNA). It is useful to removethis exonuclease proof-reading activity when using modified nucleotidesto prevent the exonuclease removing the unnatural nucleotide afterincorporation. These changes to DNA polymerase enzymes are useful forDNA sequencing applications involving 3′ modified nucleotides. Suchchanges have previously been made for the Vent and Deep Vent DNApolymerases. As described in WO 2005/024010, modifications to theso-called motif A region, amino acid positions 408-410 of 9° Npolymerases, exhibit improved incorporation of nucleotide analoguesbearing substituents at the 3′ position of the sugar. To note, aminoacids at positions 408, 409, 410 in a 9° N polymerase are functionallyequivalent to amino acids 409, 410, and 411 in wild type P. abyssi andP. horikoshii. This trio of amino acids are in close proximity to thenucleotide that is being incorporated.

For brevity, amino acid mutation nomenclature is used throughout thisapplication. One having skill in the art would understand the amino acidmutation nomenclature, such that D141A refers to aspartic acid (singleletter code is D), at position 141, is replaced with alanine (singleletter code A). Likewise, it is understood that when an amino acidmutation nomenclature is used and the terminal amino acid code ismissing, e.g., P411, it is understood that no mutation was made relativeto the wild type.

All polymerases used were expressed in E. coli BL21 STAR (DE3)(ThermoFisher) as follows. First, the gene was synthesized (ATUM) inpD451-SR expression vector using codons selected for high-yieldexpression. Whenever needed, additional in vitro substitutionmutagenesis was performed on the gene sequence using a Site DirectedMutagenesis Kit (New England Biolabs) and confirmed by Sangersequencing. Clones for each variant were transformed into BL21 STAR(DE3) (ThermoFisher) and incubated at 37° C. with shaking in 2.0 Lflasks until an OD₆₀₀ of 0.6 was reached. Then, isopropylβ-d-1-thiogalactopyranoside (IPTG) (1 mM final concentration) was addedto induce specific protein expression. The cells were incubated for 3hours at 37° C. and collected by centrifugation at 5000 rpm for 5minutes. Cell pellets were stored at minus 80° C.

All purification steps were performed at 4° C. except as indicated. Thefrozen cell paste was resuspended in lysis buffer (20 mM Tris pH 7.5,500 mM NaCl, 1 mM EDTA, 1 mM DTT, 10% Glycerol, 1 mM PMSF, and proteaseinhibitor cocktail, EDTA-free (Thermo Fisher). The suspended cells weresubjected to sonication and centrifuged at 20,000 rpm for 20 minutes.Then, the supernatant was heated to 80° C. for 30 min to inactivate hostproteins. The mixture was again centrifuged at 20,000 rpm for 20 min andthe pellet discarded.

The supernatant was collected and Poly(ethyleneimine) was added slowlyto the supernatant to a final concentration of 0.4% with continuedstirring for 30 minutes. The mixture was centrifuged at 20,000 rpm for20 min and the pellet was discarded. Then, solid ammonium sulfate wasadded to the supernatant to 65% saturation with stirring continued for30 minutes. The mixture was centrifuged at 20,000 rpm for 20 minutes andthe precipitated proteins were resuspended in 10 ml Buffer A (20 mM TrispH 7.0, 50 mM KCl, 0.1 mM EDTA, 10% glycerol, 1 mM DTT, 1 mM PMSF). Theprotein was dialyzed overnight against 1 Liter Buffer A. The dialyzedsample was centrifuged at 20,000 rpm for 20 minutes to remove anyprecipitate and the supernatant was loaded onto a 5 ml Hi-Trap SP FFcolumn (GE Healthcare) equilibrated with Buffer A. The polymerase waseluted using a 100 ml gradient from 50 to 800 mM KCl. Peak fractionswere pooled and dialyzed overnight against Buffer B (20 mM Tris pH 7.5,50 mM KCl, 0.1 mM EDTA, 10% glycerol, 1 mM DTT, 1 mM PMSF). The dialyzedsample was centrifuged at 20,000 rpm for 20 minutes to remove anyprecipitate and the supernatant was loaded onto a 5 ml Hi-Trap Heparincolumn (GE Healthcare) equilibrated with Buffer B. The polymerase waseluted using a 100 ml gradient from 50 to 800 mM KCl. Peak fractionswere pooled and dialyzed overnight against storage buffer (20 mM Tris pH7.5, 100 mM KCl, 0.1 mM EDTA, 50% glycerol, 1 mM DTT) and stored at −20°C.

Polymerase and exonuclease assays were done using a fluorescentprimer/template as described elsewhere (see, for example, Nikiforov T T.et al. Anal Biochem. 2014 Sep. 15; 461:67-73; and Nikiforov T T. et al.Anal Biochem. 2011 May 15; 412(2):229-36). Pyrococcus horikoshii wildtype polymerase (SEQ ID NO.: 1; alternatively referred to herein asSE-1) showed both polymerase and exonuclease activity. Variants wereconstructed and tested for polymerase, exonuclease and for activity withnucleotide reversible terminators and for sequencing SBS. The fullplasmid nucleic acid sequence of wild type P. horikoshii OT3 gene clonedin plasmid used to generate the P. horikoshii protein SN00 is providedin SEQ ID NO: 2. The nucleic acid sequence for wild type P. horikoshiiOT3 gene is provided as SEQ ID NO: 3. Additional Pyrococcus species areprovided in SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24,SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO:29 and SEQ ID NO: 30.

All variants were profiled against isomeric reversible terminators.Balancing incorporation kinetics and fidelity is a challenge. Throughrational design of the motif A region, a series of mutations thataccelerate incorporation with an acceptable fidelity score wasdiscovered. The i-Term probe refers to an isomeric reversible terminatorhaving two possible isomers. For example, an i-term probe has theformula

wherein R^(A) and R^(B) are hydrogen or alkyl, wherein at least one ofR^(A) or R^(B) are hydrogen to yield a steroisomeric probe, and R^(C) isthe remainder of the reversible terminator. For the experimentsdescribed herein, the i-Term probe has two isomers, iso-1 and iso-2. Theapplicants observed that the polymerases described herein are capable ofincorporating modified nucleotides containing all four natural DNA basesA, T, C and G (and nucleotide analogues thereof).

Included herein are sequences of Pyrococcus based enzymes (e.g., P.horikoshii and P. abyssi) that are useful for nucleic acid sequencing.

Example 2: Assay for Incorporation of Nucleotide Reversible Terminators

The rate of incorporation of a fluorescent nucleotide reversibleterminator (NRT) was measured using primer/templates attached toavidin-coated magnetic beads (MyOne C1, ThermoFisher). The5′-biotinylated 160 primer is annealed to the appropriate 160-X templateand bound to the beads along with a tethering oligo5′-Biotin-CG(TAGCCG)₆TAGC-3ddC (tether B). The beads are then attachedto the surface of 384-well streptavidin-coated plates (Greiner Bio-one)to which tether A (5′-Biotin-GC(TACGGC)₆TACG-3ddC) has previously beenbound. Reactions are initiated in a house-developed buffer by theaddition of 100 nM nucleotides (or 300 nM nucleotides for Challengetemplate sequences, unless otherwise indicated) and 133 nM DNApolymerase at a temperature of 65° C. The reaction is stopped byflooding duplicate wells with room temperature wash buffer afterincubation for 15 seconds and additional wells after 10 minutes. Blankswere also made without incubation. The wells were imaged under afluorescence microscope, and the images analyzed using software thatidentifies fluorescent beads and calculates their average brightness.The blank was subtracted from the time points and the values at 15seconds and 10 minutes used to calculate the half-time of incorporationassuming first-order kinetics with completion in under 10 minutes.

TABLE 1 General Template Sequences 160-1 5′- (SEQ ID NO: 4)GACTCACATGAATCAGTGCAGCATCAGATGTATGACCGAAGCGGACGAAGG TGCGTGGA-3ddC 160-25′- (SEQ ID NO: 5) GTGGTTCATCGCGTCCGATATCAAACTTCGTCAAGTCGAAGCGGACGAAGGTGCGTGGA-3ddC 160-3 5′- (SEQ ID NO: 6)TACTAGGTTGTACGATCCCTGCACTTCAGCTAAGCACGAAGCGGACGAAGG TGCGTGGA-3ddC 160-45′- (SEQ ID NO: 7) AGCTACCAATATTTAGTTTCCGAGTCTCAGCTCATGCGAAGCGGACGAAGGTGCGTGGA-3ddC 160 Primer 5′-Biotin- (SEQ ID NO: 8)AAAAAAAAAAAAGTCCACGCACCTTCGTCCGCTTCG

The underlined nucleotide in Table 1 is the first one downstream fromthe 160 primer.

Through ongoing SBS experiments, data shows that certain nucleic acidsequences in the template that precede the nucleotide about to beincorporated can temporarily stall or slow down incorporation of thenext nucleotide. Generally, they are GC-rich sequences; for example,some difficult sequences in the template that precede nucleotide to beincorporated may be described in Table 2.

TABLE 2 Difficult sequences Difficult sequences in the Nucleotidetemplate that precede the to be complementary nucleotide incorporatedto be incorporated T 5′-CCGCC (SEQ ID NO: 17) G 5′-GCGCT (SEQ ID NO: 18)A 5′-CCGCG (SEQ ID NO: 19) C 5-ACGCC (SEQ ID NO: 20)

Therefore, a set of templates, dubbed ‘challenge-templates,’ weredevised to assist in identifying polymerase mutants capable of rapidnucleotide incorporation. An example of the challenge template sequencesare listed in Table 3, and the assay conditions are the same as theconditions used for the General Template sequences provided in Table 1.To note, the underlined sequences in the challenge-templates correspondto the difficult sequences identified in Table 2, while the boldnucleotide refers to the nucleotide complement to be incorporated.

TABLE 3 Challenge Template Sequences 260-1 5′- (SEQ ID NO: 9)CCAACTTGATATTAATAACACTATAGACCA CCGCCCGAAGCGGACGAAGGT GCGTGGA/3ddC/ 260-25′- (SEQ ID NO: 10) ATGATTAAACTCCTAAGCAGAAAACCTACC GCGCTCGAAGCGGACGAAGGTGCGTGGA/3ddC/ 260-3 5′- (SEQ ID NO: 11) TCTTTAATAACCTGATTCAGCGAAACCAATCCGCGCGAAGCGGACGAAGGT GCGTGGA/3ddC/ 260-4 5′- (SEQ ID NO: 12)CGGTTATCGCTGGCGACTCCTTCGAGATGG ACGCCCGAAGCGGACGAAGGT GCGTGGA/3ddC/260-1 Primer 52- (SEQ ID NO: 13)Bio/AAAAAAAAAAAAGTCCACGCACCTTCGTCCGCTTCGGGCGG 260-2 Primer 52-(SEQ ID NO: 14) Bio/AAAAAAAAAAAAGTCCACGCACCTTCGTCCGCTTCGAGCGC260-3 Primer 52- (SEQ ID NO: 15)Bio/AAAAAAAAAAAAGTCCACGCACCTTCGTCCGCTTCGCGCGG 260-4 Primer 52-(SEQ ID NO: 16) Bio/AAAAAAAAAAAAGTCCACGCACCTTCGTCCGCTTCGGGCGT

Example 3: Isomer Differentiation

Certain mutations in the polymerase favor the incorporation of oneisomer, thus creating optimized polymerases for a unique class ofreversible terminators.

In embodiments, the nucleotide is

wherein Base is a Base as described herein, R³ is —OH, monophosphate, orpolyphosphate or a nucleic acid, and R′ is a reversible terminatorhaving the formula:

wherein R^(A) and R^(B) are hydrogen or alkyl and R^(C) is the remainderof the reversible terminator. In embodiments, R^(A) is methyl, R^(B) ishydrogen, and R^(C) is the remainder of the reversible terminatormoiety. In embodiments, R′ has the formula

wherein R^(C) is the remainder of the reversible terminator moiety. Inembodiments, the nucleotide is

wherein the Base is cytosine or a derivative thereof (e.g., cytosineanalogue), guanine or a derivative thereof (e.g., guanine analogue),adenine or a derivative thereof (e.g., adenine analogue), thymine or aderivative thereof (e.g., thymine analogue), uracil or a derivativethereof (e.g., uracil analogue), hypoxanthine or a derivative thereof(e.g., hypoxanthine analogue), xanthine or a derivative thereof (e.g.,xanthine analogue), guanosine or a derivative thereof (e.g.,7-methylguanosine analogue), deaza-adenine or a derivative thereof(e.g., deaza-adenine analogue), deaza-guanine or a derivative thereof(e.g., deaza-guanine), deaza-hypoxanthine or a derivative thereof,5,6-dihydrouracil or a derivative thereof (e.g., 5,6-dihydrouracilanalogue), 5-methylcytosine or a derivative thereof (e.g.,5-methylcytosine analogue), or 5-hydroxymethylcytosine or a derivativethereof (e.g., 5-hydroxymethylcytosine analogue) moieties. Inembodiments, the base is thymine, cytosine, uracil, adenine, guanine,hypoxanthine, xanthine, theobromine, caffeine, uric acid, or isoguanine.

Though one parameter, the average half time of nucleotide incorporationis measured over all four nucleotides (A, T, C, and G), and serves as auseful indicator of the enzyme kinetics. Described in Table 4 is theaverage halftime, t_(1/2), averaged over each of the four incorporatednucleotides (i.e., A, T, C, and G) for halftime measurements using theGeneral templates (i.e., sequences described in Table 1) and theChallenge templates (i.e., the sequences described in Table 3). Becausethe reversible terminator i-term has two possible isomers, both thefirst isomer (iso-1) and the second isomer (iso-2) are reported in Table4. Surprisingly, variants of the polymerase displayed preference for oneisomer.

Reactions were initiated in a house-developed buffer by the addition of100 nM nucleotides (or 300 nM nucleotides for Challenge templatesequences, unless otherwise indicated) and 133 nM DNA polymerase at atemperature of 65° C. The data presented in Table 4 corresponds to thedata presented in FIGS. 1A-1C and FIG. 2.

TABLE 4 Reported average half time of incorporation of modifiednucleotides bearing reversible terminator probes iso-1 and iso-2.Internal General templates Challenge templates Ref # t_(1/2) iso-1 (s)t_(1/2) i so-2 (s) t_(1/2) iso-1 (s) t_(1/2) iso-2 (s) SE-1 30.22 130.28SE-2 9.05 15.16 SE-181 152.52 114.08 [100 nM nucs] [100 nM nucs] SE-18377.14 52.40 [100 nM nucs] [100 nM nucs] SE-3 84.67 23.42 SE-4 99.2943.08 SE-6 33.15 11.19 SE-7 111.54 54.36 SE-8 97.59 47.56 SE-9 42.2917.97 SE-10 198.59 76.97 SE-52 7.90 7.48 SE-53 10.04 11.66 SE-5 105.4144.90 SE-11 41.49 16.20 SE-12 99.11 44.08 SE-13 31.04 13.21 SE-14 135.8849.98 SE-15 132.66 85.43 SE-21 47.27 20.81 SE-20 173.4 116.88 [100 nMnucs] [100 nM nucs] SE-24 149.99 94.56 [100 nM nucs] [100 nM nucs] SE-2849.98 32.05 [100 nM nucs] [100 nM nucs] SE-31 155.55 109.19 [100 nMnucs] [100 nM nucs] SE-51 12.60 22.49 SE-56 5.28 6.73 SE-57 8.03 15.81SE-58 31.58 22.29 SE-59 21.09 22.20 SE-60 22.30 29.85 [100 nM nucs] [100nM nucs] SE-61 34.69 58.78 [100 nM nucs] [100 nM nucs] SE-68 44.27 58.78[100 nM nucs] [100 nM nucs] SE-69 39.18 44.93 [100 nM nucs] [100 nMnucs] SE-70 61.85 91.13 [100 nM nucs] [100 nM nucs] SE-71 6.60 6.22 1.966.02 SE-72 6.11 10.00 5.30 18.01 SE-73 6.57 12.71 17.03 42.92 SE-74 4.518.27 8.97 14.8 SE-75 7.98 7.88 8.24 16.74 SE-62 4.51 7.84 6.41 24.60SE-63 9.27 6.96 4.34 8.13 SE-64 7.36 7.01 3.27 8.96 SE-65 6.51 7.81 6.1819.59

Example 4: Measuring Enzyme Fidelity

The fidelity of a DNA polymerase is the result of accurate replicationof a desired template. Specifically, this involves multiple steps,including the ability to read a template strand, select the appropriatenucleoside triphosphate and insert the correct nucleotide at the 3′primer terminus, such that Watson-Crick base pairing is maintained. Inaddition to effective discrimination of correct versus incorrectnucleotide incorporation, some DNA polymerases possess a 3′→5′exonuclease activity. This activity, known as “proofreading”, is used toexcise incorrectly incorporated mononucleotides that are then replacedwith the correct nucleotide. In embodiments of the invention describedherein, the exonuclease activity has been removed, therefore it isimportant to have a high fidelity enzyme.

High-fidelity DNA polymerases have safeguards to protect against bothmaking and propagating mistakes while copying DNA. Such mutatedpolymerases have a significant binding preference for the correct versusthe incorrect nucleotide during polymerization. Fidelity of thepolymerase may be quantified using any suitable method known in the art.For example, to quantify the fidelity herein, the method includesperforming a single nucleotide extension where the next base to beincorporated is known (e.g., A) in the presence of excess incorrectnucleotide (e.g., G). For example, the enzyme, template, primercomposition is mixed with 5 mM dATP and 500 mM dGTP (the most likelymisincorporation), to probe nucleotide incorporation with 100-foldexcess of the wrong nucleotide. The reported fidelity ratio is thesignal (relative fluorescence units) from the correct base divided bythe signal from the incorrect base, multiplied by 100. Therefore, ahigher fidelity score corresponds to a lower rate of misincorporation(i.e., incorporating the incorrect nucleotide).

TABLE 5 Reported fidelity ratio for incorporation of modifiednucleotides bearing reversible terminator probes iso-1 and iso-2.Internal Fidelity Fidelity Ref # iso-1 iso-2 SE-1 1,755 970 SE-2 262 651SE-3 175 589 SE-4 705 2,324 SE-6 706 832 SE-7 1,703 2,117 SE-8 540 2,638SE-9 541 709 SE-10 1,392 2,114 SE-5 586 1,852 SE-11 475 512 SE-12 4281,673 SE-13 466 513 SE-14 1,667 1,881 SE-15 1,162 1,333 SE-231 10,41722,313 SE-232 8,933 14,182 SE-233 8,736 22,538 SE-234 12,857 26,055SE-184 2,852 5,652 SE-185 2,505 5,724 SE-188 1,435 8,818 SE-19 5,9979,186 SE-22 6,687 7,759 SE-25 5,642 6,187 SE-26 6,396 8,148

Reaction details are described elsewhere herein.

Example 5: Mutational Analysis of Pyrococcus Variants

Through site directed mutagenesis, key mutations in the motif A region(i.e., the three amino acids functionally equivalent or homologous toamino acids 409, 410, and 411 in wild type P. abyssi and P. horikoshii)were determined. Experiments in P. horikoshii and P. abyssi suggest fourgeneral classes of motif A regions (amino acid positions 409, 410, and411) that provide superior incorporation of modified nucleotidesrelative to 9N7. The first class has motif A as: SAP, SAV, SGI, AAV,SAI, SAG, or SGP, along with the amino acids 141A and 143A. The secondclass has motif A as AGI, AGP, SGI, SGP, AGG, AGV, or SGS, along withthe amino acids 129A, 141A, 143A, and 486A. The third class has motif Aas SAP, SAV, SGI, AAV, SAI, SAG, or SGP, along with the amino acids141A, 143A, and 153E. The fourth class has motif A as AGI, AGP, SGI,SGP, AGG, AGV, or SGS, along with the amino acids 129A, 141A, 143A,486A, and 153E.

To note, when amino acid position 411 is proline (or a positionfunctionally equivalent to 411), this corresponds to the wild-type aminoacid and not a mutated amino acid. As described in Seo et al. J Org Chem68(2): 609-12 (2003) and WO 2005/024010, it was widely believed thatmodifications to the motif A region are required to incorporatenucleotide analogues; and so it was surprising to find that onlymodifying positions 409 and 410 in the motif A region not only resultedin the incorporation of a modified nucleotide, but significantlyimproved incorporation kinetics. Table 6 includes additional mutantswith these motif A region modifications, as well as other modificationsto the backbone to improve DNA binding, increase fidelity, improvemodified nucleotide incorporation, and deter exonuclease activity.Interestingly, particular combinations of mutations permitdifferentiation for isomeric reversible terminators.

TABLE 6 List of mutations in variant polymerases. The mutations in thistable are mutations relative to the wild type P. horikoshii (SEQ IDNO:1). Internal Ref # Amino acids SE-1 D141A; E143A; L409S; Y410A; P411VSE-2 D141A; E143A; L409S; Y410A; P411V; A486V SE-3 M129A; D141A; E143A;L409A; Y410A; P411I; A486V SE-4 M129A; D141A; E143A; L409A; Y410G;P411I; A486V SE-5 M129A; D141A; E143A; T144A; L409A; Y410G; P411I; A486VSE-6 M129A; D141A; E143A; L409A; Y410G; P411I; A486V; T515S SE-7 M129A;D141A; E143A; L409A; Y410G; P411I; A486V; T515S; I522L SE-8 M129A;D141A; E143A; L409A; Y410G; P411I; A486V; T591I SE-9 M129A; D141A;E143A; L409A; Y410G; P411I; A486V; T515S; T591I SE-10 M129A; D141A;E143A; L409A; Y410G; P411I; A486V; T515S; I522L; T591I SE-11 M129A;D141A; E143A; T144A; L409A; Y410G; P411I; A486V; T515S SE-12 M129A;D141A; E143A; T144A; L409A; Y410G; P411I; A486V; T591I SE-13 M129A;D141A; E143A; T144A; L409A; Y410G; P411I; A486V; T515S; T591I SE-14M129A; D141A; E143A; T144A; L409A; Y410G; P411I; A486V; T515S; I522LSE-15 M129A; D141A; E143A; T144A; L409A; Y410G; P411I; A486V; T515S;I522L; T591I SE-16 M129A; D141A; E143A; L409A; Y410G; P411I; A486V;G153E SE-17 M129A; D141A; E143A; T144A; L409A; Y410G; P411I; A486V;T515S; T591I; G153E SE-18 M129A; D141A; E143A; T144A; L409A; Y410G;P411I; A486V; T515S; T591I; K713E SE-19 M129A; D141A; E143A; T144A;L409A; Y410G; P411I; A486V; T515S; T591I; K477W; K478A SE-20 M129A;D141A; E143A; T144A; L409A; Y410G; P411I; A486V; T515S; T591I; A486LSE-21 M129A; D141A; E143A; T144A; L409A; Y410G; P411I; A486V; T515S;T591I; K603A SE-22 M129A; D141A; E143A; T144A; L409A; Y410G; P411I;A486V; T515S; T591I; N736A SE-23 M129A; D141A; E143A; T144A; L409A;Y410G; P411I; A486V; T515S; T591I; K477W SE-24 M129A; D141A; E143A;T144A; L409A; Y410G; P411I; A486V; T515S; T591I; K477A SE-25 M129A;D141A; E143A; T144A; L409A; Y410G; P411I; A486V; T515S; T591I; K478ASE-26 M129A; D141A; E143A; T144A; L409A; Y410G; P411I; A486V; T515S;T591I; L479S SE-27 M129A; D141A; E143A; T144A; L409A; Y410G; P411I;A486V; T515S; T591I; K477A; K478A; L479S SE-28 M129A; D141A; E143A;T144A; L409A; Y410G; P411I; A486V; T515S; T591I; A640L SE-29 M129A;D141A; E143A; T144A; L409A; Y410G; P411I; A486V; T515S; T591I; E719ASE-30 M129A; D141A; E143A; T144A; L409A; Y410G; P411I; A486V; T515S;T591I; R714A SE-31 M129A; D141A; E143A; T144A; L409A; Y410G; P411I;A486V; T515S; T591I; D215A SE-32 M129A; D141A; E143A; T144A; L409A;Y410G; P411I; A486V; T515S; T591I; D315A SE-33 M129A; D141A; E143A;T144A; L409A; Y410G; P411I; A486V; T515S; T591I; D215A; D315A SE-34M129A; D141A; E143A; T144A; L409A; Y410G; P411I; A486V; T515S; T591I;K713E; G153E SE-35 M129A; D141A; E143A; T144A; L409A; Y410G; P411I;A486V; T515S; T591I; K477W; K478A; G153E SE-36 M129A; D141A; E143A;T144A; L409A; Y410G; P411I; A486V; T515S; T591I; A486L; G153E SE-37M129A; D141A; E143A; T144A; L409A; Y410G; P411I; A486V; T515S; T591I;K603A; G153E SE-38 M129A; D141A; E143A; T144A; L409A; Y410G; P411I;A486V; T515S; T591I; N736A; G153E SE-39 M129A; D141A; E143A; T144A;L409A; Y410G; P411I; A486V; T515S; T591I; K477W; G153E SE-40 M129A;D141A; E143A; T144A; L409A; Y410G; P411I; A486V; T515S; T591I; K477A;G153E SE-41 M129A; D141A; E143A; T144A; L409A; Y410G; P411I; A486V;T515S; T591I; K478A; G153E SE-42 M129A; D141A; E143A; T144A; L409A;Y410G; P411I; A486V; T515S; T591I; L479S; G153E SE-43 M129A; D141A;E143A; T144A; L409A; Y410G; P411I; A486V; T515S; T591I; K477A; K478A;L479S; G153E SE-44 M129A; D141A; E143A; T144A; L409A; Y410G; P411I;A486V; T515S; T591I; A640L; G153E SE-45 M129A; D141A; E143A; T144A;L409A; Y410G; P411I; A486V; T515S; T591I; E719A; G153E SE-46 M129A;D141A; E143A; T144A; L409A; Y410G; P411I; A486V; T515S; T591I; R714A;G153E SE-47 M129A; D141A; E143A; T144A; L409A; Y410G; P411I; A486V;T515S; T591I; D215A; G153E SE-48 M129A; D141A; E143A; T144A; L409A;Y410G; P411I; A486V; T515S; T591I; D315A; G153E SE-49 M129A; D141A;E143A; T144A; L409A; Y410G; P411I; A486V; T515S; T591I; D215A; D315A;G153E SE-50 M129A; D141A; E143A; L409A; Y410G; A486V SE-51 M129A; D141A;E143A; T144A; L409A; Y410G; A486V SE-52 M129A; D141A; E143A; L409A;Y410G; A486V; T515S SE-53 M129A; D141A; E143A; L409A; Y410G; A486V;T591I SE-54 M129A; D141A; E143A; L409A; Y410G; A486V; G153E SE-55 M129A;D141A; E143A; L409A; Y410G; A486V; T515S; T591I SE-56 M129A; D141A;E143A; T144A; L409A; Y410G; A486V; T515S SE-57 M129A; D141A; E143A;T144A; L409A; Y410GI; A486V; T591I SE-58 M129A; D141A; E143A; T144A;L409A; Y410G; A486V; T515S; T591I SE-59 M129A; D141A; E143A; T144A;L409A; Y410G; A486V; T515S; I522L; T591I SE-60 M129A; D141A; E143A;T144A; L409A; Y410G; A486V; T515S; T591I; G153E SE-61 M129A; D141A;E143A; T144A; L409A; Y410G; A486V; T515S; T591I; K713E SE-62 M129A;D141A; E143A; T144A; L409A; Y410G; A486V; T515S; T591I; K477W; K478ASE-63 M129A; D141A; E143A; T144A; L409A; Y410G; A486V; T515S; T591I;A486L SE-64 M129A; D141A; E143A; T144A; L409A; Y410G; A486V; T515S;T591I; K603A SE-65 M129A; D141A; E143A; T144A; L409A; Y410G; A486V;T515S; T591I; N736A SE-66 M129A; D141A; E143A; T144A; L409A; Y410G;A486V; T515S; T591I; K477W SE-67 M129A; D141A; E143A; T144A; L409A;Y410G; A486V; T515S; T591I; K477A SE-68 M129A; D141A; E143A; T144A;L409A; Y410G; A486V; T515S; T591I; K478A SE-69 M129A; D141A; E143A;T144A; L409A; Y410G; A486V; T515S; T591I; L479S SE-70 M129A; D141A;E143A; T144A; L409A; Y410G; A486V; T515S; T591I; K477A; K478A; L479SSE-71 M129A; D141A; E143A; T144A; L409A; Y410G; A486V; T515S; T591I;A640L SE-72 M129A; D141A; E143A; T144A; L409A; Y410G; A486V; T515S;T591I; E719A SE-73 M129A; D141A; E143A; T144A; L409A; Y410G; A486V;T515S; T591I; R714A SE-74 M129A; D141A; E143A; T144A; L409A; Y410G;A486V; T515S; T591I; D215A SE-75 M129A; D141A; E143A; T144A; L409A;Y410G; A486V; T515S; T591I; D315A SE-76 M129A; D141A; E143A; T144A;L409A; Y410G; A486V; T515S; T591I; D215A; D315A SE-77 M129A; D141A;E143A; T144A; L409A; Y410G; A486V; T515S; T591I; K713E; G153E SE-78M129A; D141A; E143A; T144A; L409A; Y410G; A486V; T515S; T591I; K477W;K478A; G153E SE-79 M129A; D141A; E143A; T144A; L409A; Y410G; A486V;T515S; T591I; A486L; G153E SE-80 M129A; D141A; E143A; T144A; L409A;Y410G; A486V; T515S; T591I; K603A; G153E SE-81 M129A; D141A; E143A;T144A; L409A; Y410G; A486V; T515S; T591I; N736A; G153E SE-82 M129A;D141A; E143A; T144A; L409A; Y410G; A486V; T515S; T591I; K477W; G153ESE-83 M129A; D141A; E143A; T144A; L409A; Y410G; A486V; T515S; T591I;K477A; G153E SE-84 M129A; D141A; E143A; T144A; L409A; Y410G; A486V;T515S; T591I; K478A; G153E SE-85 M129A; D141A; E143A; T144A; L409A;Y410G; A486V; T515S; T591I; L479S; G153E SE-86 M129A; D141A; E143A;T144A; L409A; Y410G; A486V; T515S; T591I; K477A; K478A; L479S; G153ESE-87 M129A; D141A; E143A; T144A; L409A; Y410G; A486V; T515S; T591I;A640L; G153E SE-88 M129A; D141A; E143A; T144A; L409A; Y410G; A486V;T515S; T591I; E719A; G153E SE-89 M129A; D141A; E143A; T144A; L409A;Y410G; A486V; T515S; T591I; R714A; G153E SE-90 M129A; D141A; E143A;T144A; L409A; Y410G; A486V; T515S; T591I; D215A; G153E SE-91 M129A;D141A; E143A; T144A; L409A; Y410G; A486V; T515S; T591I; D315A; G153ESE-92 M129A; D141A; E143A; T144A; L409A; Y410G; A486V; T515S; T591I;D215A; D315A; G153E SE-93 M129A; D141A; E143A; T144A; L409A; Y410G;A486V; T515S; T591I; K713E; A640L SE-94 M129A; D141A; E143A; T144A;L409A; Y410G; A486V; T515S; T591I; K477W; K478A; A640L SE-95 M129A;D141A; E143A; T144A; L409A; Y410G; T515S; T591I; A486L; A640L SE-96M129A; D141A; E143A; T144A; L409A; Y410G; A486V; T515S; T591I; K603A;A640L SE-97 M129A; D141A; E143A; T144A; L409A; Y410G; A486V; T515S;T591I; N736A; A640L SE-98 M129A; D141A; E143A; T144A; L409A; Y410G;A486V; T515S; T591I; K477W; A640L SE-99 M129A; D141A; E143A; T144A;L409A; Y410G; A486V; T515S; T591I; K477A; A640L SE-100 M129A; D141A;E143A; T144A; L409A; Y410G; A486V; T515S; T591I; K478A; A640L SE-101M129A; D141A; E143A; T144A; L409A; Y410G; A486V; T515S; T591I; L479S;A640L SE-102 M129A; D141A; E143A; T144A; L409A; Y410G; A486V; T515S;T591I; K477A; K478A; L479S; A640L SE-103 M129A; D141A; E143A; T144A;L409A; Y410G; A486V; T515S; T591I; E719A; A640L SE-104 M129A; D141A;E143A; T144A; L409A; Y410G; A486V; T515S; T591I; R714A; A640L SE-105M129A; D141A; E143A; T144A; L409A; Y410G; A486V; T515S; T591I; D215A;A640L SE-106 M129A; D141A; E143A; T144A; L409A; Y410G; A486V; T515S;T591I; D315A; A640L SE-107 M129A; D141A; E143A; T144A; L409A; Y410G;A486V; T515S; T591I; D215A; D315A; A640L SE-108 M129A; D141A; E143A;T144A; L409A; Y410G; T515S; T591I; K713E; A486L SE-109 M129A; D141A;E143A; T144A; L409A; Y410G; T515S; T591I; K477W; K478A; A486L SE-110M129A; D141A; E143A; T144A; L409A; Y410G; T515S; T591I; K603A; A486LSE-111 M129A; D141A; E143A; T144A; L409A; Y410G; T515S; T591I; N736A;A486L SE-112 M129A; D141A; E143A; T144A; L409A; Y410G; T515S; T591I;K477W; A486L SE-113 M129A; D141A; E143A; T144A; L409A; Y410G; T515S;T591I; K477A; A486L SE-114 M129A; D141A; E143A; T144A; L409A; Y410G;T515S; T591I; K478A; A486L SE-115 M129A; D141A; E143A; T144A; L409A;Y410G; T515S; T591I; L479S; A486L SE-116 M129A; D141A; E143A; T144A;L409A; Y410G; T515S; T591I; K477A; K478A; L479S; A486L SE-117 M129A;D141A; E143A; T144A; L409A; Y410G; T515S; T591I; E719A; A486L SE-118M129A; D141A; E143A; T144A; L409A; Y410G; T515S; T591I; R714A; A486LSE-119 M129A; D141A; E143A; T144A; L409A; Y410G; T515S; T591I; D215A;A486L SE-120 M129A; D141A; E143A; T144A; L409A; Y410G; T515S; T591I;D315A; A486L SE-121 M129A; D141A; E143A; T144A; L409A; Y410G; T515S;T591I; D215A; D315A; A486L SE-122 M129A; D141A; E143A; T144A; L409A;Y410G; A486V; T515S; T591I; K477W; K478AK; K713E SE-123 M129A; D141A;E143A; T144A; L409A; Y410G; A486V; T515S; T591I; K603A; K713E SE-124M129A; D141A; E143A; T144A; L409A; Y410G; A486V; T515S; T591I; N736A;K713E SE-125 M129A; D141A; E143A; T144A; L409A; Y410G; A486V; T515S;T591I; K477W; K713E SE-126 M129A; D141A; E143A; T144A; L409A; Y410G;A486V; T515S; T591I; K477A; K713E SE-127 M129A; D141A; E143A; T144A;L409A; Y410G; A486V; T515S; T591I; K478A; K713E SE-128 M129A; D141A;E143A; T144A; L409A; Y410G; A486V; T515S; T591I; L479S; K713E SE-129M129A; D141A; E143A; T144A; L409A; Y410G; A486V; T515S; T591I; K477A;K478A; L479S; K713E SE-130 M129A; D141A; E143A; T144A; L409A; Y410G;A486V; T515S; T591I; E719A; K713E SE-131 M129A; D141A; E143A; T144A;L409A; Y410G; A486V; T515S; T591I; R714A; K713E SE-132 M129A; D141A;E143A; T144A; L409A; Y410G; A486V; T515S; T591I; D215A; K713E SE-133M129A; D141A; E143A; T144A; L409A; Y410G; A486V; T515S; T591I; D315A;K713E SE-134 M129A; D141A; E143A; T144A; L409A; Y410G; A486V; T515S;T591I; D215A; D315A; K713E SE-135 M129A; D141A; E143A; T144A; L409A;Y410G; A486V; T515S; T591I; K477W; K478A; K603A SE-136 M129A; D141A;E143A; T144A; L409A; Y410G; A486V; T515S; T591I; N736A; K603A SE-137M129A; D141A; E143A; T144A; L409A; Y410G; A486V; T515S; T591I; K477W;K603A SE-138 M129A; D141A; E143A; T144A; L409A; Y410G; A486V; T515S;T591I; K477A; K603A SE-139 M129A; D141A; E143A; T144A; L409A; Y410G;A486V; T515S; T591I; K478A; K603A SE-140 M129A; D141A; E143A; T144A;L409A; Y410G; A486V; T515S; T591I; L479S; K603A SE-141 M129A; D141A;E143A; T144A; L409A; Y410G; A486V; T515S; T591I; K477A; K478A; L479S;K603A SE-142 M129A; D141A; E143A; T144A; L409A; Y410G; A486V; T515S;T591I; E719A; K603A SE-143 M129A; D141A; E143A; T144A; L409A; Y410G;A486V; T515S; T591I; R714A; K603A SE-144 M129A; D141A; E143A; T144A;L409A; Y410G; A486V; T515S; T591I; D215A; K603A SE-145 M129A; D141A;E143A; T144A; L409A; Y410G; A486V; T515S; T591I; D315A; K603A SE-146M129A; D141A; E143A; T144A; L409A; Y410G; A486V; T515S; T591I; D215A;D315A; K603A SE-147 M129A; D141A; E143A; L409A; Y410G; A486V; T515S;G153 SE-148 M129A; D141A; E143A; L409A; Y410G; A486V; T515S; K713ESE-149 M129A; D141A; E143A; L409A; Y410G; A486V; T515S; K477W; K478ASE-150 M129A; D141A; E143A; L409A; Y410G; A486V; T515S; A486L SE-151M129A; D141A; E143A; L409A; Y410G; A486V; T515S; K603A SE-152 M129A;D141A; E143A; L409A; Y410G; A486V; T515S; N736A SE-153 M129A; D141A;E143A; L409A; Y410G; A486V; T515S; K477W SE-154 M129A; D141A; E143A;L409A; Y410G; A486V; T515S; K477A SE-155 M129A; D141A; E143A; L409A;Y410G; A486V; T515S; L478A SE-156 M129A; D141A; E143A; L409A; Y410G;A486V; T515S; L479S SE-157 M129A; D141A; E143A; L409A; Y410G; A486V;T515S; K477A; K478A; L479S SE-158 M129A; D141A; E143A; L409A; Y410G;A486V; T515S; A640L SE-159 M129A; D141A; E143A; L409A; Y410G; A486V;T515S; E719A SE-160 M129A; D141A; E143A; L409A; Y410G; A486V; T515S;R714A SE-161 M129A; D141A; E143A; L409A; Y410G; A486V; T515S; D215ASE-162 M129A; D141A; E143A; L409A; Y410G; A486V; T515S; D315A SE-163M129A; D141A; E143A; L409A; Y410G; A486V; T515S; D215A; D315A SE-164M129A; D141A; E143A; L409A; Y410G; A486V; T591I; G153 SE-165 M129A;D141A; E143A; L409A; Y410G; A486V; T591I; K713E SE-166 M129A; D141A;E143A; L409A; Y410G; A486V; T591I; K477W; K478A SE-167 M129A; D141A;E143A; L409A; Y410G; A486V; T591I; A486L SE-168 M129A; D141A; E143A;L409A; Y410G; A486V; T591I; K603A SE-169 M129A; D141A; E143A; L409A;Y410G; A486V; T591I; N736A SE-170 M129A; D141A; E143A; L409A; Y410G;A486V; T591I; K477W SE-171 M129A; D141A; E143A; L409A; Y410G; A486V;T591I; K477A SE-172 M129A; D141A; E143A; L409A; Y410G; A486V; T591I;L478A SE-173 M129A; D141A; E143A; L409A; Y410G; A486V; T591I; L479SSE-174 M129A; D141A; E143A; L409A; Y410G; A486V; T591I; K477A; K478A;L479S SE-175 M129A; D141A; E143A; L409A; Y410G; A486V; T591I; A640LSE-176 M129A; D141A; E143A; L409A; Y410G; A486V; T591I; E719A SE-177M129A; D141A; E143A; L409A; Y410G; A486V; T591I; R714A SE-178 M129A;D141A; E143A; L409A; Y410G; A486V; T591I; D215A SE-179 M129A; D141A;E143A; L409A; Y410G; A486V; T591I; D315A SE-180 M129A; D141A; E143A;L409A; Y410G; A486V; T591I; D215A; D315A SE-181 D141A; E143A; L409S;Y410A; P411V; A486L SE-182 D141A; E143A; L409S; Y410A; P411V; A486V;T515S SE-183 D141A; E143A; L409S; Y410A; P411V; A486V; T591I SE-184D141A; E143A; T144A; ; L409S; Y410A; P411V; A486V SE-185 D141A; E143A;L409S; Y410A; P411V; A486V; G153E SE-186 D141A; E143A; L409S; Y410A;P411V; A486V; T144A; G153E SE-187 D141A; E143A; L409S; Y410A; P411V;A486V; K477W SE-188 D141A; E143A; L409S; Y410A; A486V SE-189 D141A;E143A; L409S; Y410A; A486L SE-190 D141A; E143A; L409S; Y410A; A486V;T515S SE-191 D141A; E143A; L409S; Y410A; A486V; T591I SE-192 D141A;E143A; T144A; ; L409S; Y410A; A486V SE-193 D141A; E143A; L409S; Y410A;A486V; G153E SE-194 D141A; E143A; L409S; Y410A; A486V; T144A; G153ESE-195 D141A; E143A; L409S; Y410A; A486V; K477W SE-196 D141A; E143A;L409S; Y410A; A486V; T515S; G153E SE-197 D141A; E143A; L409S; Y410A;A486V; T515S; K713E SE-198 D141A; E143A; L409S; Y410A; A486V; T515S;K477W; K478A SE-199 D141A; E143A; L409S; Y410A; A486L; T515S SE-200D141A; E143A; L409S; Y410A; A486V; T515S; K603A SE-201 D141A; E143A;L409S; Y410A; A486V; T515S; N736A SE-202 D141A; E143A; L409S; Y410A;A486V; T515S; K477W SE-203 D141A; E143A; L409S; Y410A; A486V; T515S;K477A SE-204 D141A; E143A; L409S; Y410A; A486V; T515S; K478A SE-205D141A; E143A; L409S; Y410A; A486V; T515S; L479S SE-206 D141A; E143A;L409S; Y410A; A486V; T515S; K477A; K478A; L479S SE-207 D141A; E143A;L409S; Y410A; A486V; T515S; A640L SE-208 D141A; E143A; L409S; Y410A;A486V; T515S; E719A SE-209 D141A; E143A; L409S; Y410A; A486V; T515S;R714A SE-210 D141A; E143A; L409S; Y410A; A486V; T515S; D215A SE-211D141A; E143A; L409S; Y410A; A486V; T515S; D315A SE-212 D141A; E143A;L409S; Y410A; A486V; T515S; D215A; D315A SE-213 D141A; E143A; L409S;Y410A; A486V; T591I; G153E SE-214 D141A; E143A; L409S; Y410A; A486V;T591I; K713E SE-215 D141A; E143A; L409S; Y410A; A486V; T591I; K477W;K478A SE-216 D141A; E143A; L409S; Y410A; A486L; T591I SE-217 D141A;E143A; L409S; Y410A; A486V; T591I; K603A SE-218 D141A; E143A; L409S;Y410A; A486V; T591I; N736A SE-219 D141A; E143A; L409S; Y410A; A486V;T591I; K477W SE-220 D141A; E143A; L409S; Y410A; A486V; T591I; K477ASE-221 D141A; E143A; L409S; Y410A; A486V; T591I; K478A SE-222 D141A;E143A; L409S; Y410A; A486V; T591I; L479S SE-223 D141A; E143A; L409S;Y410A; A486V; T591I; K477A; K478A; L479S SE-224 D141A; E143A; L409S;Y410A; A486V; T591I; A640L SE-225 D141A; E143A; L409S; Y410A; A486V;T591I; E719A SE-226 D141A; E143A; L409S; Y410A; A486V; T591I; R714ASE-227 D141A; E143A; L409S; Y410A; A486V; T591I; D215A SE-228 D141A;E143A; L409S; Y410A; A486V; T591I; D315A SE-229 D141A; E143A; L409S;Y410A; A486V; T591I; D215A; D315A SE-230 D141A; E143A; L409S; Y410A;A486V; T591I; G153E; T515S SE-231 M129A; D141A; E143A; T144A; L409A;Y410G; P411I; A486V; T515S; T591I; G153E; K713E; K478A SE-232 M129A;D141A; E143A; T144A; L409A; Y410G; P411I; A486V; T515S; T591I; G153E;K713E; L479S SE-233 M129A; D141A; E143A; T144A; L409A; Y410G; P411I;A486V; T515S; T591I; G153E; K713E; K477A; K478A; L479S SE-234 M129A;D141A; E143A; T144A; L409A; Y410G; P411I; A486V; T515S; T591I; G153E;K713E; A640L SE-235 M129A; D141A; E143A; L409S; Y410G; A486V SE-236M129A; D141A; E143A; T144A; L409S; Y410G; A486V SE-237 M129A; D141A;E143A; L409S; Y410G; A486V; T515S SE-238 M129A; D141A; E143A; L409S;Y410G; A486V; T591I SE-239 M129A; D141A; E143A; L409S; Y410G; A486V;G153E SE-240 M129A; D141A; E143A; L409S; Y410G; A486V; T515S; T591ISE-241 M129A; D141A; E143A; T144A; L409S; Y410G; A486V; T515S SE-242M129A; D141A; E143A; T144A; L409S; Y410GI; A486V; T591I SE-243 M129A;D141A; E143A; T144A; L409S; Y410G; A486V; T515S; T591I SE-244 M129A;D141A; E143A; T144A; L409S; Y410G; A486V; T515S; I522L; T591I SE-245M129A; D141A; E143A; T144A; L409S; Y410G; A486V; T515S; T591I; G153ESE-246 M129A; D141A; E143A; T144A; L409S; Y410G; A486V; T515S; T591I;K713E SE-247 M129A; D141A; E143A; T144A; L409S; Y410G; A486V; T515S;T591I; K477W; K478A SE-248 M129A; D141A; E143A; T144A; L409S; Y410G;A486L; T515S; T591I SE-249 M129A; D141A; E143A; T144A; L409S; Y410G;A486V; T515S; T591I; K603A SE-250 M129A; D141A; E143A; T144A; L409S;Y410G; A486V; T515S; T591I; N736A SE-251 M129A; D141A; E143A; T144A;L409S; Y410G; A486V; T515S; T591I; K477W SE-252 M129A; D141A; E143A;T144A; L409S; Y410G; A486V; T515S; T591I; K477A SE-253 M129A; D141A;E143A; T144A; L409S; Y410G; A486V; T515S; T591I; K478A SE-254 M129A;D141A; E143A; T144A; L409S; Y410G; A486V; T515S; T591I; L479S SE-255M129A; D141A; E143A; T144A; L409S; Y410G; A486V; T515S; T591I; K477A;K478A; L479S SE-256 M129A; D141A; E143A; T144A; L409S; Y410G; A486V;T515S; T591I; A640L SE-257 M129A; D141A; E143A; T144A; L409S; Y410G;A486V; T515S; T591I; E719A SE-258 M129A; D141A; E143A; T144A; L409S;Y410G; A486V; T515S; T591I; R714A SE-259 M129A; D141A; E143A; T144A;L409S; Y410G; A486V; T515S; T591I; D215A SE-260 M129A; D141A; E143A;T144A; L409S; Y410G; A486V; T515S; T591I; D315A SE-261 M129A; D141A;E143A; T144A; L409S; Y410G; A486V; T515S; T591I; D215A; D315A SE-262M129A; D141A; E143A; T144A; L409S; Y410G; A486V; T515S; T591I; K713E;G153E SE-263 M129A; D141A; E143A; T144A; L409S; Y410G; A486V; T515S;T591I; K477W; K478A; G153E SE-264 M129A; D141A; E143A; T144A; L409S;Y410G; A486V; T515S; T591I; A486L; G153E SE-265 M129A; D141A; E143A;T144A; L409S; Y410G; A486V; T515S; T591I; K603A; G153E SE-266 M129A;D141A; E143A; T144A; L409S; Y410G; A486V; T515S; T591I; N736A; G153ESE-267 M129A; D141A; E143A; T144A; L409S; Y410G; A486V; T515S; T591I;K477W; G153E SE-268 M129A; D141A; E143A; T144A; L409S; Y410G; A486V;T515S; T591I; K477A; G153E SE-269 M129A; D141A; E143A; T144A; L409S;Y410G; A486V; T515S; T591I; K478A; G153E SE-270 M129A; D141A; E143A;T144A; L409S; Y410G; A486V; T515S; T591I; L479S; G153E SE-271 M129A;D141A; E143A; T144A; L409S; Y410G; A486V; T515S; T591I; K477A; K478A;L479S; G153E SE-272 M129A; D141A; E143A; T144A; L409S; Y410G; A486V;T515S; T591I; A640L; G153E SE-273 M129A; D141A; E143A; T144A; L409S;Y410G; A486V; T515S; T591I; E719A; G153E SE-274 M129A; D141A; E143A;T144A; L409S; Y410G; A486V; T515S; T591I; R714A; G153E SE-275 M129A;D141A; E143A; T144A; L409S; Y410G; A486V; T515S; T591I; D215A; G153ESE-276 M129A; D141A; E143A; T144A; L409S; Y410G; A486V; T515S; T591I;D315A; G153E SE-277 M129A; D141A; E143A; T144A; L409S; Y410G; A486V;T515S; T591I; D215A; D315A; G153E SE-278 M129A; D141A; E143A; T144A;L409S; Y410G; A486V; T515S; T591I; K713E; A640L SE-279 M129A; D141A;E143A; T144A; L409S; Y410G; A486V; T515S; T591I; K477W; K478A; A640LSE-280 M129A; D141A; E143A; T144A; L409S; Y410G; T515S; T591I; A486LA640L SE-281 M129A; D141A; E143A; T144A; L409S; Y410G; A486V; T515S;T591I; K603A; A640L SE-282 M129A; D141A; E143A; T144A; L409S; Y410G;A486V; T515S; T591I; N736A; A640L SE-283 M129A; D141A; E143A; T144A;L409S; Y410G; A486V; T515S; T591I; K477W; A640L SE-284 M129A; D141A;E143A; T144A; L409S; Y410G; A486V; T515S; T591I; K477A; A640L SE-285M129A; D141A; E143A; T144A; L409S; Y410G; A486V; T515S; T591I; K478A;A640L SE-286 M129A; D141A; E143A; T144A; L409S; Y410G; A486V; T515S;T591I; L479S; A640L SE-287 M129A; D141A; E143A; T144A; L409S; Y410G;A486V; T515S; T591I; K477A; K478A; L479S; A640L SE-288 M129A; D141A;E143A; T144A; L409S; Y410G; A486V; T515S; T591I; E719A; A640L SE-289M129A; D141A; E143A; T144A; L409S; Y410G; A486V; T515S; T591I; R714A;A640L SE-290 M129A; D141A; E143A; T144A; L409S; Y410G; A486V; T515S;T591I; D215A; A640L SE-291 M129A; D141A; E143A; T144A; L409S; Y410G;A486V; T515S; T591I; D315A; A640L SE-292 M129A; D141A; E143A; T144A;L409S; Y410G; A486V; T515S; T591I; D215A; D315A; A640L SE-293 M129A;D141A; E143A; T144A; L409S; Y410G; T515S; T591I; K713E; A486L SE-294M129A; D141A; E143A; T144A; L409S; Y410G; T515S; T591I; K477W; K478A;A486L SE-295 M129A; D141A; E143A; T144A; L409S; Y410G; T515S; T591I;K603A; A486L SE-296 M129A; D141A; E143A; T144A; L409S; Y410G; T515S;T591I; N736A; A486L SE-297 M129A; D141A; E143A; T144A; L409S; Y410G;T515S; T591I; K477W; A486L SE-298 M129A; D141A; E143A; T144A; L409S;Y410G; T515S; T591I; K477A; A486L SE-299 M129A; D141A; E143A; T144A;L409S; Y410G; T515S; T591I; K478A; A486L SE-300 M129A; D141A; E143A;T144A; L409S; Y410G; T515S; T591I; L479S; A486L SE-301 M129A; D141A;E143A; T144A; L409S; Y410G; T515S; T591I; K477A; K478A; L479S; A486LSE-302 M129A; D141A; E143A; T144A; L409S; Y410G; T515S; T591I; E719A;A486L SE-303 M129A; D141A; E143A; T144A; L409S; Y410G; T515S; T591I;R714A; A486L SE-304 M129A; D141A; E143A; T144A; L409S; Y410G; T515S;T591I; D215A; A486L SE-305 M129A; D141A; E143A; T144A; L409S; Y410G;T515S; T591I; D315A; A486L SE-306 M129A; D141A; E143A; T144A; L409S;Y410G; T515S; T591I; D215A; D315A; A486L SE-307 M129A; D141A; E143A;T144A; L409S; Y410G; A486V; T515S; T591I; K477W; K478AK; K713E SE-308M129A; D141A; E143A; T144A; L409S; Y410G; A486V; T515S; T591I; K603A;K713E SE-309 M129A; D141A; E143A; T144A; L409S; Y410G; A486V; T515S;T591I; N736A; K713E SE-310 M129A; D141A; E143A; T144A; L409S; Y410G;A486V; T515S; T591I; K477W; K713E SE-311 M129A; D141A; E143A; T144A;L409S; Y410G; A486V; T515S; T591I; K477A; K713E SE-312 M129A; D141A;E143A; T144A; L409S; Y410G; A486V; T515S; T591I; K478A; K713E SE-313M129A; D141A; E143A; T144A; L409S; Y410G; A486V; T515S; T591I; L479S;K713E SE-314 M129A; D141A; E143A; T144A; L409S; Y410G; A486V; T515S;T591I; K477A; K478A; L479S; K713E SE-315 M129A; D141A; E143A; T144A;L409S; Y410G; A486V; T515S; T591I; E719A; K713E SE-316 M129A; D141A;E143A; T144A; L409S; Y410G; A486V; T515S; T591I; R714A; K713E SE-317M129A; D141A; E143A; T144A; L409S; Y410G; A486V; T515S; T591I; D215A;K713E SE-318 M129A; D141A; E143A; T144A; L409S; Y410G; A486V; T515S;T591I; D315A; K713E SE-319 M129A; D141A; E143A; T144A; L409S; Y410G;A486V; T515S; T591I; D215A; D315A; K713E SE-320 M129A; D141A; E143A;T144A; L409S; Y410G; A486V; T515S; T591I; K477W; K478A; K603A SE-321M129A; D141A; E143A; T144A; L409S; Y410G; A486V; T515S; T591I; N736A;K603A SE-322 M129A; D141A; E143A; T144A; L409S; Y410G; A486V; T515S;T591I; K477W; K603A SE-323 M129A; D141A; E143A; T144A; L409S; Y410G;A486V; T515S; T591I; K477A; K603A SE-324 M129A; D141A; E143A; T144A;L409S; Y410G; A486V; T515S; T591I; K478A; K603A SE-325 M129A; D141A;E143A; T144A; L409S; Y410G; A486V; T515S; T591I; L479S; K603A SE-326M129A; D141A; E143A; T144A; L409S; Y410G; A486V; T515S; T591I; K477A;K478A; L479S; K603A SE-327 M129A; D141A; E143A; T144A; L409S; Y410G;A486V; T515S; T591I; E719A; K603A SE-328 M129A; D141A; E143A; T144A;L409S; Y410G; A486V; T515S; T591I; R714A; K603A SE-329 M129A; D141A;E143A; T144A; L409S; Y410G; A486V; T515S; T591I; D215A; K603A SE-330M129A; D141A; E143A; T144A; L409S; Y410G; A486V; T515S; T591I; D315A;K603A SE-331 M129A; D141A; E143A; T144A; L409S; Y410G; A486V; T515S;T591I; D215A; D315A; K603A SE-332 M129A; D141A; E143A; L409S; Y410G;A486V; T515S; G153 SE-333 M129A; D141A; E143A; L409S; Y410G; A486V;T515S; K713E SE-334 M129A; D141A; E143A; L409S; Y410G; A486V; T515S;K477W; K478A SE-335 M129A; D141A; E143A; L409S; Y410G; A486V; T515S;A486L SE-336 M129A; D141A; E143A; L409S; Y410G; A486V; T515S; K603ASE-337 M129A; D141A; E143A; L409S; Y410G; A486V; T515S; N736A SE-338M129A; D141A; E143A; L409S; Y410G; A486V; T515S; K477W SE-339 M129A;D141A; E143A; L409S; Y410G; A486V; T515S; K477A SE-340 M129A; D141A;E143A; L409S; Y410G; A486V; T515S; L478A SE-341 M129A; D141A; E143A;L409S; Y410G; A486V; T515S; L479S SE-342 M129A; D141A; E143A; L409S;Y410G; A486V; T515S; K477A; K478A; L479S SE-343 M129A; D141A; E143A;L409S; Y410G; A486V; T515S; A640L SE-344 M129A; D141A; E143A; L409S;Y410G; A486V; T515S; E719A SE-345 M129A; D141A; E143A; L409S; Y410G;A486V; T515S; R714A SE-346 M129A; D141A; E143A; L409S; Y410G; A486V;T515S; D215A SE-347 M129A; D141A; E143A; L409S; Y410G; A486V; T515S;D315A SE-348 M129A; D141A; E143A; L409S; Y410G; A486V; T515S; D215A;D315A SE-349 M129A; D141A; E143A; L409S; Y410G; A486V; T591I; G153SE-350 M129A; D141A; E143A; L409S; Y410G; A486V; T591I; K713E SE-351M129A; D141A; E143A; L409S; Y410G; A486V; T591I; K477W; K478A SE-352M129A; D141A; E143A; L409S; Y410G; A486V; T591I; A486L SE-353 M129A;D141A; E143A; L409S; Y410G; A486V; T591I; K603A SE-354 M129A; D141A;E143A; L409S; Y410G; A486V; T591I; N736A SE-355 M129A; D141A; E143A;L409S; Y410G; A486V; T591I; K477W SE-356 M129A; D141A; E143A; L409S;Y410G; A486V; T591I; K477A SE-357 M129A; D141A; E143A; L409S; Y410G;A486V; T591I; L478A SE-358 M129A; D141A; E143A; L409S; Y410G; A486V;T591I; L479S SE-359 M129A; D141A; E143A; L409S; Y410G; A486V; T591I;K477A; K478A; L479S SE-360 M129A; D141A; E143A; L409S; Y410G; A486V;T591I; A640L SE-361 M129A; D141A; E143A; L409S; Y410G; A486V; T591I;E719A SE-362 M129A; D141A; E143A; L409S; Y410G; A486V; T591I; R714ASE-363 M129A; D141A; E143A; L409S; Y410G; A486V; T591I; D215A SE-364M129A; D141A; E143A; L409S; Y410G; A486V; T591I; D315A SE-365 M129A;D141A; E143A; L409S; Y410G; A486V; T591I; D215A; D315A SE-366 D141A;E143A; L409A; Y410G; A486V; T515S; G153E SE-367 D141A; E143A; L409A;Y410G; A486V; T515S; K477W; K478A SE-368 D141A; E143A; L409A; Y410G;A486V; T515S; A640L SE-369 D141A; E143A; L409A; Y410G; A486V; T515S;L479S SE-370 D141A; E143A; L409A; Y410G; A486V; T515S; G153E; K477W;K478A SE-371 D141A; E143A; L409A; Y410G; A486V; T515S; G153E; A640LSE-372 D141A; E143A; L409A; Y410G; A486V; T515S; G153E; L479S SE-373D141A; E143A; L409A; Y410G; A486V; T515S; G153E; K477W; K478A; L479SSE-374 D141A; E143A; L409A; Y410G; A486V; T515S; G153E; K477W; K478A;A640L SE-375 D141A; E143A; L409A; Y410G; A486V; T515S; G153E; K477W;K478A; L479S; A640L SE-376 D141A; E143A; L409A; Y410G; A486V; T515S;K477W; K478A; L479S SE-377 D141A; E143A; L409A; Y410G; A486V; T515S;K477W; K478A; A640L SE-378 D141A; E143A; L409A; Y410G; A486V; T515S;K477W; K478A; L479S; A640L SE-379 D141A; E143A; L409A; Y410G; A486V;T515S; K479S; A640L SE-380 D141A; E143A; L409A; Y410G; A486V; T515S;K479S; A640L; G153E SE-381 M129A; D141A; E143A; T144A; L409A; Y410G;A486V; T515S; T591I; K477W; K478A; N736A SE-382 M129A; D141A; E143A;T144A; L409A; Y410G; A486V; T515S; T591I; K477W; K478A; L479S SE-383M129A; D141A; E143A; T144A; L409A; Y410G; A486V; T515S; T591I; K477W;K478A; E719A SE-384 M129A; D141A; E143A; T144A; L409A; Y410G; A486V;T515S; T591I; K477W; K478A; R714A SE-385 M129A; D141A; E143A; T144A;L409A; Y410G; A486V; T515S; T591I; K477W; K478A; D215A SE-386 M129A;D141A; E143A; T144A; L409A; Y410G; A486V; T515S; T591I; K477W; K478A;D315A SE-387 M129A; D141A; E143A; T144A; L409A; Y410G; A486V; T515S;T591I; K477W; K478A; D215A; D315A SE-388 M129A; D141A; E143A; T144A;L409A; Y410G; A486V; T515S; T591I; L479S; N736A SE-389 M129A; D141A;E143A; T144A; L409A; Y410G; A486V; T515S; T591I; L479S; K477W SE-390M129A; D141A; E143A; T144A; L409A; Y410G; A486V; T515S; T591I; L479S;K477A SE-391 M129A; D141A; E143A; T144A; L409A; Y410G; A486V; T515S;T591I; L479S; K478A SE-392 M129A; D141A; E143A; T144A; L409A; Y410G;A486V; T515S; T591I; L479S; E719A SE-393 M129A; D141A; E143A; T144A;L409A; Y410G; A486V; T515S; T591I; L479S; R714A SE-394 M129A; D141A;E143A; T144A; L409A; Y410G; A486V; T515S; T591I; L479SD215A SE-395M129A; D141A; E143A; T144A; L409A; Y410G; A486V; T515S; T591I; L479S;D315A SE-396 M129A; D141A; E143A; T144A; L409A; Y410G; A486V; T515S;T591I; L479S; D215A; D315A SE-397 M129A; D141A; E143A; T144A; L409A;Y410G; A486V; T515S; T591I; K477W; K478A; G153E; L479S SE-398 M129A;D141A; E143A; T144A; L409A; Y410G; A486V; T515S; T591I; K477W; K478A;G153E; A640L SE-399 M129A; D141A; E143A; T144A; L409A; Y410G; A486V;T515S; T591I; K477W; K478A; G153E; L479S; A640L SE-400 M129A; D141A;E143A; T144A; L409A; Y410G; A486V; T515S; T591I; G153E; L479S; A640LSE-401 M129A; D141A; E143A; T144A; L409A; Y410G; A486V; T515S; T591I;K477W; K478A; A640L; L479S

Example 6: Mutational Analysis of Pyrococcus Species

The information gained by examining Pyrococcus horikoshii and Pyrococcusabyssi may be translated to additional Pyrococcus species. Included inTable 7 are additional Pyrococcus species and the proposed mutations.The table is in condensed form, rather than showing every particularcombination of mutations, however it is understood that the table isintended to be exhaustive. For example, the first species is Pyrococcuswoesei. The motif A amino acids may be SAV, SAP, SGI, AAV, SAI, SAG, orSGP (i.e., amino acid position 409 is S, amino acid position 410 is A,and amino acid position 411 is V). This particular motif A region (SAV)may be combined with one or more additional possible mutations disclosedin the third column (e.g., M129A, T144A, and/or R714A).

TABLE 7 Pyrococcus species and amino acid selections to enableincorporation of modified nucleotides. The mutations are relative to theparent sequence (i.e., the sequence indicated in the first column).Motif A (amino acid positions 408, 409, Additional mutations Pyrococcusspecies and 410) (individual, or combined) Pyrococcus woisei SAV, SAP,SGI, AAV, Motif A and one or more of (SEQ ID NO:22) SAI, SAG, or SGP thefollowing: M129A; D141A; D143A; T144A; G153E; D215A; D315A; D215A andD315A; K477W; K477A; I478A; L479S; K477W and I478A; K477A and I478A andL479S; A486V; A486L; T515S; T591I; K603A; A640L; N713E; R714A; E719A;E720A; N736A Pyrococcus furiosus SAV, SAP, SGI, AAV, Motif A and one ormore of (SEQ ID NO:23) SAI, SAG, or SGP the following: M129A; D141A;D143A; T144A; G153E; D215A; D315A; D215A and D315A; K477W; K477A; I478A;L479S; K477W and I478A; K477A and I478A and L479S; A486V; A486L; T515S;T591I; K603A; A640L; N713E; R714A; E719A; E720A; N736A Pyrococcusglycovarans SAV, SAP, SGI, AAV, Motif A and one or more of (SEQ IDNO:24) SAI, SAG, or SGP the following: M129A; D141A; D143A; T144A;A153E; D215A; D315A; D215A and D315A; K477W; K477A; K478A; M479S; M479L;K477W/K478A; K477A and K478A and M479S; K477A and K478A and M479L;A486V; A486L; T515S; T591I; K603A; A640L; N713E; R714A; E719A; E720A;N736A; Pyrococcus sp. NA2 SAV, SAP, SGI, AAV, Motif A and one or more of(SEQ ID NO:25) SAI, SAG, or SGP the following M129A; D141A; D143A;T144A; G153E; D215A; D315A; D215A and D315A; R477W; R477A; K478A; L479S;R477W and K478A; R477A and K478A and L479S; A486V; A486L; T515S; T591I;K603A; A640L; K713E; R714A; E719A; E720A; N736A; Pyrococcus sp. ST700;note SAV, SAP, SGI, AAV, , Motif A and one or more of the sequence isnaturally SAI SAG, or SGP the following M129A; missing 16 amino acidsfrom D141A; D143A; T144A; the C-terminus G153E; D215A; D315A; (SEQ IDNO:26) D215A/D315A; K477W; K477A; K478A; L479S; K477W and K478A; K477Aand K478A and L479S; A486V; A486L; T515S; T591I; K603A; A640L; K713E;R714A; E719A; E720A; N736A Pyrococcus kukulkanii SAV, SAP, SGI, AAV,Motif A and one or more of (SEQ ID NO:27) SAI, SAG, or SGP the followingM129A; D141A; D143A; T144A; A153E; D215A; D315A; D215A and D315A; K477W;K477A; K478A; M479S; M479L; K477W and K478A; K477A and K478A and M479S;K477A and K478A and M479L; A486V; A486L; T515S; T591I; K603A; A640L;K713E; R714A; E719A; E720A; N736A; Pyrococcus yayanosii SAV, SAP, SGI,AAV, Motif A and one or more of (SEQ ID NO:28) SAI, SAG, or SGP thefollowing M129A; D141A; D143A; T144A; G153E; D215A; D315A; D215A andD315A; R477W; R477A; K478A; L479S; R477W and K478A; R477A andK478A/L4795; A486V; A486L; T515S; T591I; K603A; A640L; R713E; R714A;E719A; E720A; N736A; Pyrococcus sp. ST04 SAV, SAP, SGI, AAV, Motif A andone or more of (SEQ ID NO:29) SAI, SAG, or SGP the following M129A;D141A; D143A; T144A; G153E; D215A; D315A; D215A and D315A; K477W; K477A;K478A; L479S; K477W and K478A; K477A and K478A and L479S; A486V; A486L;T515S; T591I; K603A; A640L; K713E; R714A; E719A; E720A; N736A;Pyrococcus sp. GB-D SAV, SAP, SGI, AAV, Motif A and one or more of (SEQID NO:30) SAI, SAG, or SGP the following M129A; D141A; D143A; T144A;A153E; D215A; D315A; D215A and D315A; K477W; K477A; K478A; M479S; K477Wand K478A; K477A and K478A and M479S; A486V; A486L; T515S; T591I; K603A;A640L; K713E; R714A; E719A; E720A; N736A;

SUMMARY

The mutations described herein can be categorized into four classes ofmutations.

Class 1: A Pyrococcus polymerase comprising an amino acid sequence thatis at least 80% identical to wild type P. horikoshii; comprising thefollowing amino acids: an alanine at amino acid position 141 or anyamino acid that is functionally equivalent to the amino acid position141; an alanine at amino acid position 143 or any amino acid that isfunctionally equivalent to the amino acid position 143; an alanine orserine at amino acid position 409 or any amino acid that is functionallyequivalent to the amino acid position 409; an alanine or glycine atamino acid position 410 or any amino acid that is functionallyequivalent to the amino acid position 410; and a valine, proline,isoleucine, or glycine at amino acid position 411 or any amino acid thatis functionally equivalent to the amino acid position 411.

Class 2: A Pyrococcus polymerase comprising an amino acid sequence thatis at least 80% identical to wild type P. horikoshii; comprising thefollowing amino acids: an alanine at amino acid position 129 or anyamino acid that is functionally equivalent to the amino acid position141; an alanine at amino acid position 141 or any amino acid that isfunctionally equivalent to the amino acid position 141; an alanine atamino acid position 143 or any amino acid that is functionallyequivalent to the amino acid position 143; an alanine or serine at aminoacid position 409 or any amino acid that is functionally equivalent tothe amino acid position 409; a glycine at amino acid position 410 or anyamino acid that is functionally equivalent to the amino acid position410; a serine, valine, proline, isoleucine, or glycine at amino acidposition 411 or any amino acid that is functionally equivalent to theamino acid position 411; and an alanine at amino acid position 486 orany amino acid that is functionally equivalent to the amino acidposition 486.

Class 3: A Pyrococcus polymerase comprising an amino acid sequence thatis at least 80% identical to wild type P. horikoshii; comprising thefollowing amino acids: an alanine at amino acid position 141 or anyamino acid that is functionally equivalent to the amino acid position141; an alanine at amino acid position 143 or any amino acid that isfunctionally equivalent to the amino acid position 143; a glutamic acidat amino acid position 153 or any amino acid that is functionallyequivalent to the amino acid position 153; an alanine or serine at aminoacid position 409 or any amino acid that is functionally equivalent tothe amino acid position 409; an alanine or glycine at amino acidposition 410 or any amino acid that is functionally equivalent to theamino acid position 410; and a valine, proline, isoleucine, or glycineat amino acid position 411 or any amino acid that is functionallyequivalent to the amino acid position 411.

Class 4: A Pyrococcus polymerase comprising an amino acid sequence thatis at least 80% identical to wild type P. horikoshii; comprising thefollowing amino acids: an alanine at amino acid position 129 or anyamino acid that is functionally equivalent to the amino acid position141; an alanine at amino acid position 141 or any amino acid that isfunctionally equivalent to the amino acid position 141; an alanine atamino acid position 143 or any amino acid that is functionallyequivalent to the amino acid position 143; a glutamic acid at amino acidposition 153 or any amino acid that is functionally equivalent to theamino acid position 153; an alanine or serine at amino acid position 409or any amino acid that is functionally equivalent to the amino acidposition 409; a glycine at amino acid position 410 or any amino acidthat is functionally equivalent to the amino acid position 410; aserine, valine, proline, isoleucine, or glycine at amino acid position411 or any amino acid that is functionally equivalent to the amino acidposition 411; and an alanine at amino acid position 486 or any aminoacid that is functionally equivalent to the amino acid position 486.

The class of mutations can be listed as follows:

Class 1 Pyrococcus specific amino acids relative to Pyrococcushorikoshii OT3 DNA polymerase wild type. (Class 1-1) D141A; E143A;L409S; Y410A; P411V. (Class 1-2) D141A; E143A; L409S; Y410A; P411P.(Class 1-3) D141A; E143A; L409S; Y410G; P411I. (Class 1-4) D141A; E143A;L409A; Y410A; P411V. (Class 1-5) D141A; E143A; L409S; Y410A; P411I.(Class 1-6) D141A; E143A; L409S; Y410A; P411G. (Class 1-7) D141A; E143A;L409S; Y410G; P411P. Additional class 1 mutations include: T144A; G153E;D215A; D315A; D215A and D315A; T515S; I522L; T591I; K477W; K477A; K478A;L479S; K477W and K478A; K477A and K478A and L479S; A486V; A486L; K603A;A640L; K713E; R714A; E719A; E720A; or N736A.

Class 2 Pyrococcus specific amino acids relative to Pyrococcushorikoshii OT3 DNA polymerase wild type. (Class 2-1) M129A; D141A;E143A; L409A; Y410G; P411I; A486A. (Class 2-2) M129A; D141A; E143A;L409A; Y410G; P411P; A486A. (Class 2-3) M129A; D141A; E143A; L409S;Y410G; P411I; A486A. (Class 2-4) M129A; D141A; E143A; L409S; Y410G;P411P; A486A. (Class 2-5) M129A; D141A; E143A; L409A; Y410G; P411G;A486A. (Class 2-6) M129A; D141A; E143A; L409A; Y410G; P411V; A486A.(Class 2-7) M129A; D141A; E143A; L409S; Y410G; P411S; A486A. Additionalclass 2 mutations include: T144A; G153E; D215A; D315A; D215A and D315A;T515S; I522L; T591I; K477W; K477A; K478A; L479S; K477W and K478A; K477Aand K478A and L479S; K603A; A640L; K713E; R714A; E719A; E720A; or N736A.

Class 3 Pyrococcus specific amino acids relative to Pyrococcushorikoshii OT3 DNA polymerase wild type: (Class 3-1) D141A; E143A;G153E; L409S; Y410A; P411P. (Class 3-2) D141A; E143A; G153E; L409S;Y410A; P411V. (Class 3-3) D141A; E143A; G153E; L409S; Y410G; P411I.(Class 3-4) D141A; E143A; G153E; L409A; Y410A; P411V. (Class 3-5) D141A;E143A; G153E; L409S; Y410A; P411I. (Class 3-5) D141A; E143A; G153E;L409S; Y410A; P411G. (Class 3-6) D141A; E143A; G153E; L409S; Y410G;P411P. Additional class 3 mutations include: T144A; D215A; D315A; D215Aand D315A; T515S; I522L; T591I; K477W; K477A; K478A; L479S; K477W andK478A; K477A and K478A and L479S; K603A; A640L; K713E; R714A; E719A;E720A; or N736A.

Class 4 Pyrococcus specific amino acids relative to Pyrococcushorikoshii OT3 DNA polymerase wild type: (Class 4-1) M129A; D141A;E143A; G153E; L409A; Y410G; P411I; A486A. (Class 4-2) M129A; D141A;E143A; G153E; L409A; Y410G; P411P; A486A. (Class 4-3) M129A; D141A;E143A; G153E; L409S; Y410G; P411I; A486A. (Class 4-4) M129A; D141A;E143A; G153E; L409S; Y410G; P411P; A486A. (Class 4-5) M129A; D141A;E143A; G153E; L409A; Y410G; P411G; A486A. (Class 4-6) M129A; D141A;E143A; G153E; L409A; Y410G; P411V; A486A. (Class 4-7) M129A; D141A;E143A; G153E; L409S; Y410G; P411S; A486A. Additional class 4 mutationsinclude: T144A; D215A; D315A; D215A and D315A; T515S; I522L; T591I;K477W; K477A; K478A; L479S; K477W and K478A; K477A and K478A and L479S;K603A; A640L; K713E; R714A; E719A; E720A; or N736A.

EMBODIMENTS

Embodiment 1-1. A polymerase comprising an amino acid sequence that isat least 80% identical to a continuous 500 amino acid sequence withinSEQ ID NO: 1; comprising the following amino acids: an alanine or serineat amino acid position 409 or an amino acid position functionallyequivalent to amino acid position 409; a glycine or alanine at aminoacid position 410 or any amino acid that is functionally equivalent toamino acid position 410; a proline, valine, glycine, or isoleucine atamino acid position 411 or any amino acid that is functionallyequivalent to amino acid position 411; and an alanine at amino acidposition 141 or an amino acid position functionally equivalent to aminoacid position 141; and an alanine at amino acid position 143 or an aminoacid position functionally equivalent to amino acid position.

Embodiment 1-2. The polymerase of Embodiment 1-1, further comprising atleast one of the following the following amino acids: an alanine atamino acid position 144 or an amino acid position functionallyequivalent to amino acid position 144; a glutamic at amino acid position153 or an amino acid position functionally equivalent to amino acidposition 153; an alanine at amino acid position 215 or an amino acidposition functionally equivalent to amino acid position 215; an alanineat amino acid position 315 or an amino acid position functionallyequivalent to amino acid position 315; an alanine at amino acidpositions 215 and 315 or amino acid positions functionally equivalent toamino acid positions 215 and 315; a serine at amino acid position 515 oran amino acid position functionally equivalent to amino acid position515; a leucine at amino acid position 522 or an amino acid positionfunctionally equivalent to amino acid position 522; an isoleucine atamino acid position 591 or an amino acid position functionallyequivalent to amino acid position 591; a tryptophan at amino acidposition 477 or an amino acid position functionally equivalent to aminoacid position 477; an alanine at amino acid position 477 or an aminoacid position functionally equivalent to amino acid position 477; analanine at amino acid position 478 or an amino acid positionfunctionally equivalent to amino acid position 478; a serine at aminoacid position 479 or an amino acid position functionally equivalent toamino acid position 479; a tryptophan at amino acid position 477 or anamino acid position functionally equivalent to amino acid position 477and an alanine at amino acid position 478 or an amino acid positionfunctionally equivalent to amino acid position 478; an alanine at aminoacid position 477 or an amino acid position functionally equivalent toamino acid position 477, an alanine at amino acid position 478 or anamino acid position functionally equivalent to amino acid position 478and a serine at amino acid position 479 or an amino acid positionfunctionally equivalent to amino acid position 479; a valine at aminoacid position 486 or an amino acid position functionally equivalent toamino acid position 486; a leucine at amino acid position 486 or anamino acid position functionally equivalent to amino acid position 486;an alanine at amino acid position 603 or an amino acid positionfunctionally equivalent to amino acid position 603; a leucine at aminoacid position 640 or an amino acid position functionally equivalent toamino acid position 640; a glutamic acid at amino acid position 713 oran amino acid position functionally equivalent to amino acid position713; an alanine at amino acid position 714 or an amino acid positionfunctionally equivalent to amino acid position 714; an alanine at aminoacid position 719 or an amino acid position functionally equivalent toamino acid position 719; an alanine at amino acid position 720 or anamino acid position functionally equivalent to amino acid position 720;or an alanine at amino acid position 736 or an amino acid positionfunctionally equivalent to amino acid position 736.

Embodiment 1-3. A polymerase comprising an amino acid sequence that isat least 80% identical to a continuous 500 amino acid sequence withinSEQ ID NO: 1; comprising the following amino acids: an alanine or serineat amino acid position 409 or an amino acid functionally equivalent toamino acid position 409; a glycine at amino acid position 410 or anamino acid functionally equivalent to amino acid position 410; aproline, valine, glycine, isoleucine, or serine at amino acid position411 or an amino acid functionally equivalent to amino acid position 411;an alanine at amino acid position 129 or an amino acid functionallyequivalent to amino acid position 129; an alanine at amino acid position141 or an amino acid functionally equivalent to amino acid position 141;an alanine at amino acid position 143 or an amino acid functionallyequivalent to amino acid position 143; and an alanine at amino acidposition 486 or an amino acid functionally equivalent to amino acidposition 486.

Embodiment 1-4. The polymerase of Embodiment 1-3, further comprising atleast one of the following amino acids: an alanine at amino acidposition 144 or an amino acid position functionally equivalent to aminoacid position 144; a glutamic at amino acid position 153 or an aminoacid position functionally equivalent to amino acid position 153; analanine at amino acid position 215 or an amino acid positionfunctionally equivalent to amino acid position 215; an alanine at aminoacid position 315 or an amino acid position functionally equivalent toamino acid position 315; an alanine at amino acid positions 215 and 315or amino acid positions functionally equivalent to amino acid positions215 and 315; a serine at amino acid position 515 or an amino acidposition functionally equivalent to amino acid position 515; a leucineat amino acid position 522 or an amino acid position functionallyequivalent to amino acid position 522; an isoleucine at amino acidposition 591 or an amino acid position functionally equivalent to aminoacid position 591; a tryptophan at amino acid position 477 or an aminoacid position functionally equivalent to amino acid position 477; analanine at amino acid position 477 or an amino acid positionfunctionally equivalent to amino acid position 477; an alanine at aminoacid position 478 or an amino acid position functionally equivalent toamino acid position 478; a serine at amino acid position 479 or an aminoacid position functionally equivalent to amino acid position 479; atryptophan at amino acid position 477 or an amino acid positionfunctionally equivalent to amino acid position 477 and an alanine atamino acid position 478 or an amino acid position functionallyequivalent to amino acid position 478; an alanine at amino acid position477 or an amino acid position functionally equivalent to amino acidposition 477, an alanine at amino acid position 478 or an amino acidposition functionally equivalent to amino acid position 478 and a serineat amino acid position 479 or an amino acid position functionallyequivalent to amino acid position 479; an alanine at amino acid position603 or an amino acid position functionally equivalent to amino acidposition 603; a leucine at amino acid position 640 or an amino acidposition functionally equivalent to amino acid position 640; a glutamicacid at amino acid position 713 or an amino acid position functionallyequivalent to amino acid position 713; an alanine at amino acid position714 or an amino acid position functionally equivalent to amino acidposition 714; an alanine at amino acid position 719 or an amino acidposition functionally equivalent to amino acid position 719; an alanineat amino acid position 720 or an amino acid position functionallyequivalent to amino acid position 720; or an alanine at amino acidposition 736 or an amino acid position functionally equivalent to aminoacid position 736.

Embodiment 1-5. A polymerase comprising an amino acid sequence that isat least 80% identical to a continuous 500 amino acid sequence withinSEQ ID NO: 1; comprising the following amino acids: an alanine or serineat amino acid position 409 or an amino acid position functionallyequivalent to amino acid position 409; a glycine or alanine at aminoacid position 410 or an amino acid position functionally equivalent toamino acid position 410; a proline, valine, glycine, or isoleucine atamino acid position 411 or an amino acid position functionallyequivalent to amino acid position 411; an alanine at amino acid position141 or an amino acid position functionally equivalent to amino acidposition 141; an alanine at amino acid position 143 or an amino acidposition functionally equivalent to amino acid position 143; and aglutamic acid at amino acid position 153 or an amino acid positionfunctionally equivalent to amino acid position 153.

Embodiment 1-6. The polymerase of Embodiment 1-5, further comprising atleast one of the following amino acids: an alanine at amino acidposition 144 or an amino acid position functionally equivalent to aminoacid position 144; an alanine at amino acid position 215 or an aminoacid position functionally equivalent to amino acid position 215; analanine at amino acid position 315 or an amino acid positionfunctionally equivalent to amino acid position 315; an alanine at aminoacid positions 215 and 315 or amino acid positions functionallyequivalent to amino acid positions 215 and 315; a serine at amino acidposition 515 or an amino acid position functionally equivalent to aminoacid position 515; a leucine at amino acid position 522 or an amino acidposition functionally equivalent to amino acid position 522; anisoleucine at amino acid position 591 or an amino acid positionfunctionally equivalent to amino acid position 591; a tryptophan atamino acid position 477 or an amino acid position functionallyequivalent to amino acid position 477; an alanine at amino acid position477 or an amino acid position functionally equivalent to amino acidposition 477; an alanine at amino acid position 478 or an amino acidposition functionally equivalent to amino acid position 478; a serine atamino acid position 479 or an amino acid position functionallyequivalent to amino acid position 479; a tryptophan at amino acidposition 477 or an amino acid position functionally equivalent to aminoacid position 477 and an alanine at amino acid position 478 or an aminoacid position functionally equivalent to amino acid position 478; analanine at amino acid position 477 or an amino acid positionfunctionally equivalent to amino acid position 477, an alanine at aminoacid position 478 or an amino acid position functionally equivalent toamino acid position 478 and a serine at amino acid position 479 or anamino acid position functionally equivalent to amino acid position 479;an alanine at amino acid position 603 or an amino acid positionfunctionally equivalent to amino acid position 603; a leucine at aminoacid position 640 or an amino acid position functionally equivalent toamino acid position 640; a glutamic acid at amino acid position 713 oran amino acid position functionally equivalent to amino acid position713; an alanine at amino acid position 714 or an amino acid positionfunctionally equivalent to amino acid position 714; an alanine at aminoacid position 719 or an amino acid position functionally equivalent toamino acid position 719; an alanine at amino acid position 720 or anamino acid position functionally equivalent to amino acid position 720;or an alanine at amino acid position 736 or an amino acid positionfunctionally equivalent to amino acid position 736.

Embodiment 1-7. A polymerase comprising an amino acid sequence that isat least 80% identical to a continuous 500 amino acid sequence withinSEQ ID NO: 1; comprising the following amino acids: an alanine or serineat amino acid position 409 or an amino acid position functionallyequivalent to amino acid position 409; a glycine at amino acid position410 or an amino acid position functionally equivalent to amino acidposition 410; a proline, valine, glycine, isoleucine, or serine at aminoacid position 411 or an amino acid position functionally equivalent toamino acid position 411; and an alanine at amino acid position 129 or anamino acid functionally equivalent to amino acid position 129; analanine at amino acid position 141 or an amino acid functionallyequivalent to amino acid position 141; an alanine at amino acid position143 or an amino acid functionally equivalent to amino acid position 143;a glutamic acid at amino acid position 153 or an amino acid positionfunctionally equivalent to amino acid position 153; and an alanine atamino acid position 486 or an amino acid functionally equivalent toamino acid position 486.

Embodiment 1-8. The polymerase of Embodiment 1-7, further comprising atleast one of the following amino acids: an alanine at amino acidposition 144 or an amino acid position functionally equivalent to aminoacid position 144; an alanine at amino acid position 215 or an aminoacid position functionally equivalent to amino acid position 215; analanine at amino acid position 315 or an amino acid positionfunctionally equivalent to amino acid position 315; an alanine at aminoacid positions 215 and 315 or amino acid positions functionallyequivalent to amino acid positions 215 and 315; a serine at amino acidposition 515 or an amino acid position functionally equivalent to aminoacid position 515; a leucine at amino acid position 522 or an amino acidposition functionally equivalent to amino acid position 522; anisoleucine at amino acid position 591 or an amino acid positionfunctionally equivalent to amino acid position 591; a tryptophan atamino acid position 477 or an amino acid position functionallyequivalent to amino acid position 477; an alanine at amino acid position477 or an amino acid position functionally equivalent to amino acidposition 477; an alanine at amino acid position 478 or an amino acidposition functionally equivalent to amino acid position 478; a serine atamino acid position 479 or an amino acid position functionallyequivalent to amino acid position 479; a tryptophan at amino acidposition 477 or an amino acid position functionally equivalent to aminoacid position 477 and an alanine at amino acid position 478 or an aminoacid position functionally equivalent to amino acid position 478; analanine at amino acid position 477 or an amino acid positionfunctionally equivalent to amino acid position 477, an alanine at aminoacid position 478 or an amino acid position functionally equivalent toamino acid position 478 and a serine at amino acid position 479 or anamino acid position functionally equivalent to amino acid position 479;an alanine at amino acid position 603 or an amino acid positionfunctionally equivalent to amino acid position 603; a leucine at aminoacid position 640 or an amino acid position functionally equivalent toamino acid position 640; a glutamic acid at amino acid position 713 oran amino acid position functionally equivalent to amino acid position713; an alanine at amino acid position 714 or an amino acid positionfunctionally equivalent to amino acid position 714; an alanine at aminoacid position 719 or an amino acid position functionally equivalent toamino acid position 719; an alanine at amino acid position 720 or anamino acid position functionally equivalent to amino acid position 720;or an alanine at amino acid position 736 or an amino acid positionfunctionally equivalent to amino acid position 736.

Embodiment 1-9. The polymerase of any one of the preceding Embodiments,wherein the polymerase comprises an amino acid sequence that is at least90% identical to a continuous 500 amino acid sequence within SEQ ID NO:1.

Embodiment 1-10. The polymerase of anyone of the preceding Embodiments,which exhibits an increased rate of incorporation of modifiednucleotides, relative to a polymerase comprising the amino acid sequenceof SEQ ID NO: 31.

Embodiment 1-11. The polymerase of any one of the preceding Embodiments,wherein the polymerase is selected from a a Pyrococcus abyssi,Pyrococcus endeavori, Pyrococcus furiosus, Pyrococcus glycovorans,Pyrococcus horikoshii, Pyrococcus kukulkanii, Pyrococcus woesei,Pyrococcus yayanosii, Pyrococcus sp., Pyrococcus sp. 1211, Pyrococcussp. 121, Pyrococcus sp. 303, Pyrococcus sp. 304, Pyrococcus sp. 312,Pyrococcus sp. 32-4, Pyrococcus sp. 321, Pyrococcus sp. 322, Pyrococcussp. 323, Pyrococcus sp. 324, Pyrococcus sp. 95-12-1, Pyrococcus sp. AV5,Pyrococcus sp. Ax99-7, Pyrococcus sp. C2, Pyrococcus sp. EX2, Pyrococcussp. Fla95-Pc, Pyrococcus sp. GB-3A, Pyrococcus sp. GB-D, Pyrococcus sp.GBD, Pyrococcus sp. GI-H, Pyrococcus sp. GI-J, Pyrococcus sp. GIL,Pyrococcus sp. HT3, Pyrococcus sp. JT1, Pyrococcus sp. LMO-A29,Pyrococcus sp. LMO-A30, Pyrococcus sp. LMO-A31, Pyrococcus sp. LMO-A32,Pyrococcus sp. LMO-A33, Pyrococcus sp. LMO-A34, Pyrococcus sp. LMO-A35,Pyrococcus sp. LMO-A36, Pyrococcus sp. LMO-A37, Pyrococcus sp. LMO-A38,Pyrococcus sp. LMO-A39, Pyrococcus sp. LMO-A40, Pyrococcus sp. LMO-A41,Pyrococcus sp. LMO-A42, Pyrococcus sp. M24D13, Pyrococcus sp. MA2.31,Pyrococcus sp. MA2.32, Pyrococcus sp. MA2.34, Pyrococcus sp. MV1019,Pyrococcus sp. MV4, Pyrococcus sp. MV7, Pyrococcus sp. MZ14, Pyrococcussp. MZ4, Pyrococcus sp. NA2, Pyrococcus sp. NS102-T, Pyrococcus sp.P12.1, Pyrococcus sp. Pikanate 5017, Pyrococcus sp. PK 5017, Pyrococcussp. ST4, Pyrococcus sp. ST700, Pyrococcus sp. Tc-2-70, Pyrococcus sp.Tc95-7C-I, Pyrococcus sp. TC95-7C-S, Pyrococcus sp. Tc95_6, Pyrococcussp. V211, Pyrococcus sp. V212, Pyrococcus sp. V221, Pyrococcus sp. V222,Pyrococcus sp. V231, Pyrococcus sp. V232, Pyrococcus sp. V61, Pyrococcussp. V62, Pyrococcus sp. V63, Pyrococcus sp. V72, Pyrococcus sp. V73,Pyrococcus sp. VB112, Pyrococcus sp. VB113, Pyrococcus sp. VB81,Pyrococcus sp. VB82, Pyrococcus sp. VB83, Pyrococcus sp. VB85,Pyrococcus sp. VB86, Pyrococcus sp. VB93 polymerase, Pyrococcus furiosusDSM 3638, Pyrococcus sp. GE23, Pyrococcus sp. GI-H, Pyrococcus sp. NA2,Pyrococcus sp. ST04, or Pyrococcus sp. ST700 polymerase.

Embodiment 1-12. The polymerase of any one of the preceding Embodiments,wherein the polymerase is Pyrococcus abyssi or Pyrococcus horikoshiipolymerase.

Embodiment 1-13. The polymerase of any one of the preceding Embodiments,which is capable of incorporating modified nucleotides at reactiontemperatures across the range of 40° C. to 80° C.

Embodiment 1-14. A method of incorporating a modified nucleotide into anucleic acid sequence comprising allowing the following components tointeract: (i) a DNA template, (ii) a nucleotide solution, and (iii) apolymerase, wherein the polymerase is a polymerase of any one ofEmbodiments 1-1 to 1-13.

Embodiment 1-15. The method of Embodiment 1-14, wherein the polymeraseis capable of incorporating a modified nucleotide into a nucleic acidsequence in stringent hybridization conditions.

Embodiment 1-16. The method of Embodiment 1-14 or 1-15, wherein thepolymerase is capable of incorporating a modified nucleotide into anucleic acid sequence at 55 to 80 degrees Celsius.

Embodiment-2

Embodiment 2-1. A polymerase comprising an amino acid sequence that isat least 80% identical to a continuous 500 amino acid sequence withinSEQ ID NO: 1; comprising the following amino acids: an alanine or serineat amino acid position 409 or an amino acid functionally equivalent toamino acid position 409; a glycine at amino acid position 410 or anamino acid functionally equivalent to amino acid position 410; aproline, valine, glycine, isoleucine, or serine at amino acid position411 or an amino acid functionally equivalent to amino acid position 411;an alanine at amino acid position 129 or an amino acid functionallyequivalent to amino acid position 129; an alanine at amino acid position141 or an amino acid functionally equivalent to amino acid position 141;an alanine at amino acid position 143 or an amino acid functionallyequivalent to amino acid position 143; and an alanine at amino acidposition 486 or an amino acid functionally equivalent to amino acidposition 486.

Embodiment 2-2. The polymerase of Embodiment 2-1, comprising thefollowing amino acids: an alanine or serine at amino acid position 409or an amino acid functionally equivalent to amino acid position 409; aglycine at amino acid position 410 or an amino acid functionallyequivalent to amino acid position 410; and a proline at amino acidposition 411 or an amino acid functionally equivalent to amino acidposition 411.

Embodiment 2-3. The polymerase of Embodiment 2-2, comprising thefollowing amino acids: an alanine at amino acid position 409 or an aminoacid functionally equivalent to amino acid position 409.

Embodiment 2-4. The polymerase of any one of Embodiments 2-1 to 2-3,further comprising a glutamic at amino acid position 153 or an aminoacid position functionally equivalent to amino acid position 153.

Embodiment 2-5. A polymerase comprising an amino acid sequence that isat least 80% identical to a continuous 500 amino acid sequence withinSEQ ID NO: 1; comprising the following amino acids: a serine at aminoacid position 409 or an amino acid position functionally equivalent toamino acid position 409; an alanine at amino acid position 410 or anyamino acid that is functionally equivalent to amino acid position 410; aproline at amino acid position 411 or any amino acid that isfunctionally equivalent to amino acid position 411; and an alanine atamino acid position 141 or an amino acid position functionallyequivalent to amino acid position 141; and an alanine at amino acidposition 143 or an amino acid position functionally equivalent to aminoacid position.

Embodiment 2-6. The polymerase of any one of Embodiments 2-2 to 2-5,further comprising at least one of the following amino acids: an alanineat amino acid position 144 or an amino acid position functionallyequivalent to amino acid position 144; a glutamic at amino acid position153 or an amino acid position functionally equivalent to amino acidposition 153; an alanine at amino acid position 215 or an amino acidposition functionally equivalent to amino acid position 215; an alanineat amino acid position 315 or an amino acid position functionallyequivalent to amino acid position 315; an alanine at amino acidpositions 215 and 315 or amino acid positions functionally equivalent toamino acid positions 215 and 315; a serine at amino acid position 515 oran amino acid position functionally equivalent to amino acid position515; a leucine at amino acid position 522 or an amino acid positionfunctionally equivalent to amino acid position 522; an isoleucine atamino acid position 591 or an amino acid position functionallyequivalent to amino acid position 591; a tryptophan at amino acidposition 477 or an amino acid position functionally equivalent to aminoacid position 477; an alanine at amino acid position 477 or an aminoacid position functionally equivalent to amino acid position 477; analanine at amino acid position 478 or an amino acid positionfunctionally equivalent to amino acid position 478; a serine at aminoacid position 479 or an amino acid position functionally equivalent toamino acid position 479; a tryptophan at amino acid position 477 or anamino acid position functionally equivalent to amino acid position 477and an alanine at amino acid position 478 or an amino acid positionfunctionally equivalent to amino acid position 478; an alanine at aminoacid position 477 or an amino acid position functionally equivalent toamino acid position 477, an alanine at amino acid position 478 or anamino acid position functionally equivalent to amino acid position 478and a serine at amino acid position 479 or an amino acid positionfunctionally equivalent to amino acid position 479; an alanine at aminoacid position 603 or an amino acid position functionally equivalent toamino acid position 603; a leucine at amino acid position 640 or anamino acid position functionally equivalent to amino acid position 640;a glutamic acid at amino acid position 713 or an amino acid positionfunctionally equivalent to amino acid position 713; an alanine at aminoacid position 714 or an amino acid position functionally equivalent toamino acid position 714; an alanine at amino acid position 719 or anamino acid position functionally equivalent to amino acid position 719;an alanine at amino acid position 720 or an amino acid positionfunctionally equivalent to amino acid position 720; or an alanine atamino acid position 736 or an amino acid position functionallyequivalent to amino acid position 736.

Embodiment 2-7. The polymerase of any one of Embodiments 2-1 to 2-5,further comprising at least one of the following: an alanine at aminoacid position 144 or an amino acid position functionally equivalent toamino acid position 144; a serine at amino acid position 515 or an aminoacid position functionally equivalent to amino acid position 515; anisoleucine at amino acid position 591 or an amino acid positionfunctionally equivalent to amino acid position 591; an alanine at aminoacid position 603 or an amino acid position functionally equivalent toamino acid position 603; a leucine at amino acid position 640 or anamino acid position functionally equivalent to amino acid position 640;or a glutamic acid at amino acid position 713 or an amino acid positionfunctionally equivalent to amino acid position 713.

Embodiment 2-8. The polymerase of any one of Embodiments 2-1 to 2-5,further comprising at least one of the following: an alanine at aminoacid position 144 or an amino acid position functionally equivalent toamino acid position 144; a serine at amino acid position 515 or an aminoacid position functionally equivalent to amino acid position 515; anisoleucine at amino acid position 591 or an amino acid positionfunctionally equivalent to amino acid position 591; or a leucine atamino acid position 640 or an amino acid position functionallyequivalent to amino acid position 640.

Embodiment 2-9. The polymerase of any one of Embodiments 2-1 to 2-5,further comprising at least one of the following: an alanine at aminoacid position 144 or an amino acid position functionally equivalent toamino acid position 144; a serine at amino acid position 515 or an aminoacid position functionally equivalent to amino acid position 515; anisoleucine at amino acid position 591 or an amino acid positionfunctionally equivalent to amino acid position 591.

Embodiment 2-10. The polymerase of any one of the preceding Embodiments,wherein the polymerase comprises an amino acid sequence that is at least90% identical to a continuous 500 amino acid sequence within SEQ ID NO:1.

Embodiment 2-11. The polymerase of any one of the preceding Embodiments,which exhibits an increased rate of incorporation of modifiednucleotides, relative to a polymerase having the following mutations:D141A; E143A; L409S; Y410A; and P411V relative to SEQ ID NO:1.

Embodiment 2-12. The polymerase of any one of the preceding Embodiments,wherein the polymerase is selected from a Pyrococcus abyssi, Pyrococcusendeavori, Pyrococcus furiosus, Pyrococcus glycovorans, Pyrococcushorikoshii, Pyrococcus kukulkanii, Pyrococcus woesei, Pyrococcusyayanosii, Pyrococcus sp., Pyrococcus sp. 12/1, Pyrococcus sp. 121,Pyrococcus sp. 303, Pyrococcus sp. 304, Pyrococcus sp. 312, Pyrococcussp. 32-4, Pyrococcus sp. 321, Pyrococcus sp. 322, Pyrococcus sp. 323,Pyrococcus sp. 324, Pyrococcus sp. 95-12-1, Pyrococcus sp. AV5,Pyrococcus sp. Ax99-7, Pyrococcus sp. C2, Pyrococcus sp. EX2, Pyrococcussp. Fla95-Pc, Pyrococcus sp. GB-3A, Pyrococcus sp. GB-D, Pyrococcus sp.GBD, Pyrococcus sp. GI-H, Pyrococcus sp. GI-J, Pyrococcus sp. GIL,Pyrococcus sp. HT3, Pyrococcus sp. JT1, Pyrococcus sp. LMO-A29,Pyrococcus sp. LMO-A30, Pyrococcus sp. LMO-A31, Pyrococcus sp. LMO-A32,Pyrococcus sp. LMO-A33, Pyrococcus sp. LMO-A34, Pyrococcus sp. LMO-A35,Pyrococcus sp. LMO-A36, Pyrococcus sp. LMO-A37, Pyrococcus sp. LMO-A38,Pyrococcus sp. LMO-A39, Pyrococcus sp. LMO-A40, Pyrococcus sp. LMO-A41,Pyrococcus sp. LMO-A42, Pyrococcus sp. M24D13, Pyrococcus sp. MA2.31,Pyrococcus sp. MA2.32, Pyrococcus sp. MA2.34, Pyrococcus sp. MV1019,Pyrococcus sp. MV4, Pyrococcus sp. MV7, Pyrococcus sp. MZ14, Pyrococcussp. MZ4, Pyrococcus sp. NA2, Pyrococcus sp. NS102-T, Pyrococcus sp.P12.1, Pyrococcus sp. Pikanate 5017, Pyrococcus sp. PK 5017, Pyrococcussp. ST4, Pyrococcus sp. ST700, Pyrococcus sp. Tc-2-70, Pyrococcus sp.Tc95-7C-I, Pyrococcus sp. TC95-7C-S, Pyrococcus sp. Tc95_6, Pyrococcussp. V211, Pyrococcus sp. V212, Pyrococcus sp. V221, Pyrococcus sp. V222,Pyrococcus sp. V231, Pyrococcus sp. V232, Pyrococcus sp. V61, Pyrococcussp. V62, Pyrococcus sp. V63, Pyrococcus sp. V72, Pyrococcus sp. V73,Pyrococcus sp. VB112, Pyrococcus sp. VB113, Pyrococcus sp. VB81,Pyrococcus sp. VB82, Pyrococcus sp. VB83, Pyrococcus sp. VB85,Pyrococcus sp. VB86, Pyrococcus sp. VB93 polymerase, Pyrococcus furiosusDSM 3638, Pyrococcus sp. GE23, Pyrococcus sp. GI-H, Pyrococcus sp. NA2,Pyrococcus sp. ST04, or Pyrococcus sp. ST700 polymerase. The polymeraseof any one of the preceding any one of Embodiments 2-1 to 2-11, whereinthe polymerase is Pyrococcus abyssi or Pyrococcus horikoshii.

Embodiment 2-13. The polymerase according to any one of the precedingEmbodiments, which is capable of incorporating modified nucleotides atreaction temperatures across the range of 40° C. to 80° C.

Embodiment 2-14. A method of incorporating a modified nucleotide into anucleic acid sequence comprising allowing the following components tointeract: (i) a nucleic acid template, (ii) a nucleotide solution, and(iii) a polymerase, wherein the polymerase is a polymerase of any one ofEmbodiments 2-1 to 2-13.

Embodiment 2-15. The method of Embodiment 2-14, wherein the polymeraseis capable of incorporating a modified nucleotide into a nucleic acidsequence in stringent hybridization conditions.

Embodiment 2-16. The method of Embodiment 2-15, wherein the polymeraseis capable of incorporating a modified nucleotide into a nucleic acidsequence at 55° C. to 80° C.

Embodiment 2-17. A method of sequencing a nucleic acid sequencecomprising: a) providing a nucleic acid template with a primerhybridized to said template to form a primer-template hybridizationcomplex; b) adding a DNA polymerase and a nucleotide solution to theprimer-template hybridization complex, wherein the DNA polymerase is apolymerase of any one of Embodiments 2-1 to 2-13 and the nucleotidesolution comprises a modified nucleotide, wherein the modifiednucleotide comprises a detectable label; c) subjecting primer-templatehybridization complex to conditions which enable the polymerase toincorporate a modified nucleotide into the primer-template hybridizationcomplex to form a modified primer-template hybridization complex; and d)detecting the detectable label; thereby sequencing a nucleic acidsequence.

Embodiment 2-18. A kit comprising the polymerase of any one ofEmbodiments 2-1 to 2-13.

SEQUENCESAmino Acid Sequence of wild type P. horikoshii OT3 (SEQ ID NO: 1):MILDADYITEDGKPIIRIFKKENGEFKVEYDRNFRPYIYALLRDDSAIDEIKKITAQRHGKVVRIVETEKIQRKFLGRPIEVWKLYLEHPQDVPAIRDKIREHPAVVDIFEYDIPFAKRYLIDKGLTPMEGNEKLTFLAVDIETLYHEGEEFGKGPVIMISYADEEGAKVITWKKIDLPYVEVVSSEREMIKRLIRVIKEKDPDVIITYNGDNFDFPYLLKRAEKLGIKLLLGRDNSEPKMQKMGDSLAVEIKGRIHFDLFPVIRRTINLPTYTLEAVYEAIFGKPKEKVYADEIAKAWETGEGLERVAKYSMEDAKVTYELGREFFPMEAQLARLVGQPVWDVSRSSTGNLVEWFLLRKAYERNELAPNKPDEKEYERRLRESYEGGYVKEPEKGLWEGIVSLDFRSLYPSIIITHNVSPDTLNREGCEEYDVAPKVGHRFCKDFPGFIPSLLGQLLEERQKIKKRMKESKDPVEKKLLDYRQRAIKILANSYYGYYGYAKARWYCKECAESVTAWGRQYIDLVRRELEARGFKVLYIDTDGLYATIPGVKDWEEVKRRALEFVDYINSKLPGVLELEYEGFYARGFFVTKKKYALIDEEGKIVTRGLEIVRRDWSEIAKETQARVLEAILKHGNVEEAVKIVKDVTEKLTNYEVPPEKLVIYEQITRPINEYKAIGPHVAVAKRLMARGIKVKPGMVIGYIVLRGDGPISKRAISIEEFDPRKHKYDAEYYIENQVLPAVERILKAFGYKREDLRWQKTKQVGLGAWIKVKKSDNA Sequence of wild type P. horikoshii OT3 gene cloned in plasmid (SEQ ID NO: 2):CCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGCCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGGCGAGAGTAGGGAACTGCCAGGCATCAAACTAAGCAGAAGGCCCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTCTGTGTTGTAAAACGACGGCCAGTCTTAAGCTCGGGCCCCCTGGGCGGTTCTGATAACGAGTAATCGTTAATCCGCAAATAACGTAAAAACCCGCTTCGGCGGGTTTTTTTATGGGGGGAGTTTAGGGAAAGAGCATTTGTCAGAATATTTAAGGGCGCCTGTCACTTTGCTTGATATATGAGAATTATTTAACCTTATAAATGAGAAAAAAGCAACGCACTTTAAATAAGATACGTTGCTTTTTCGATTGATGAACACCTATAATTAAACTATTCATCTATTATTTATGATTTTTTGTATATACAATATTTCTAGTTTGTTAAAGAGAATTAAGAAAATAAATCTCGAAAATAATAAAGGGAAAATCAGTTTTTGATATCAAAATTATACATGTCAACGATAATACAAAATATAATACAAACTATAAGATGTTATCAGTATTTATTATGCATTTAGAATAAATTTTGTGTCGCCCTTCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTTTGAGACCTTAAGGAGGTAAAAAATGATTCTGGACGCTGATTATATTACTGAAGATGGTAAACCGATTATTCGTATTTTTAAAAAAGAAAATGGCGAGTTCAAAGTTGAATATGACCGTAACTTTCGTCCGTACATCTACGCGCTGTTGCGCGACGATAGCGCGATCGATGAGATTAAGAAAATTACCGCGCAGCGTCATGGTAAAGTTGTTCGCATCGTTGAAACCGAGAAAATTCAACGTAAATTCCTGGGCCGCCCAATTGAAGTGTGGAAGCTGTACCTGGAGCATCCGCAAGATGTCCCGGCGATCCGTGACAAGATTCGCGAGCACCCGGCCGTCGTCGACATTTTCGAATACGATATTCCGTTCGCAAAGCGTTACCTGATCGATAAGGGTCTGACCCCGATGGAGGGTAATGAAAAGCTGACGTTCCTGGCTGTCGATATTGAAACGTTGTACCACGAGGGTGAAGAGTTTGGTAAGGGCCCGGTCATTATGATCAGCTACGCGGATGAAGAGGGCGCCAAAGTTATCACGTGGAAAAAAATTGATCTGCCGTACGTTGAAGTTGTGTCCAGCGAGCGCGAGATGATTAAACGCTTGATTCGTGTGATTAAAGAAAAAGATCCAGACGTGATCATTACCTATAATGGTGACAACTTTGACTTTCCGTACTTGCTGAAACGTGCTGAGAAACTGGGTATCAAGCTGTTGCTGGGTCGCGATAATAGCGAGCCGAAGATGCAAAAAATGGGCGATAGCCTGGCAGTCGAGATCAAGGGTCGCATCCACTTTGATCTCTTTCCGGTGATTCGTCGCACGATCAATCTGCCGACCTATACGCTGGAAGCTGTCTACGAGGCAATCTTTGGTAAGCCGAAAGAAAAAGTCTATGCGGACGAAATTGCGAAAGCGTGGGAAACCGGCGAGGGCCTGGAGCGTGTGGCAAAGTACTCTATGGAAGATGCCAAAGTGACCTATGAACTGGGTCGTGAGTTCTTCCCAATGGAAGCCCAGTTGGCGCGCTTGGTGGGCCAACCGGTTTGGGACGTTTCCCGTAGCAGCACCGGTAACCTGGTTGAGTGGTTTCTGTTGCGTAAAGCGTATGAGCGTAATGAACTGGCACCGAACAAGCCTGACGAGAAAGAATATGAACGTCGCCTGCGTGAATCTTACGAGGGTGGTTACGTCAAAGAACCGGAAAAGGGTCTGTGGGAAGGCATCGTGAGCCTGGATTTCCGTAGCCTGTACCCTAGCATCATCATCACGCACAATGTTAGCCCGGACACCCTGAACCGCGAGGGCTGCGAAGAGTACGACGTTGCGCCGAAAGTCGGCCATCGTTTTTGTAAAGACTTCCCTGGTTTCATCCCAAGCCTGCTGGGTCAGCTGCTGGAAGAGAGACAGAAAATTAAAAAACGCATGAAAGAATCGAAAGATCCGGTTGAGAAAAAGCTGCTGGATTACCGCCAGCGTGCCATCAAGATTCTGGCTAACTCATATTATGGCTACTACGGTTATGCTAAAGCGCGTTGGTACTGTAAAGAGTGCGCGGAGTCCGTCACCGCGTGGGGTCGCCAGTATATCGATCTGGTGCGTCGCGAGCTGGAAGCGCGTGGTTTTAAGGTCCTGTACATCGATACTGACGGTCTGTATGCAACCATCCCTGGTGTCAAAGACTGGGAAGAGGTTAAGCGTCGTGCACTGGAATTTGTGGACTATATCAATTCTAAGTTGCCGGGTGTGCTGGAGCTGGAGTACGAAGGCTTCTATGCACGCGGCTTTTTCGTTACGAAAAAGAAATACGCACTGATCGACGAAGAGGGCAAGATTGTGACTCGTGGTCTGGAAATCGTTCGTCGCGACTGGAGCGAGATTGCAAAAGAAACCCAAGCTCGCGTTCTGGAAGCAATCCTGAAACATGGTAACGTCGAAGAAGCCGTCAAGATCGTGAAAGATGTCACCGAAAAGTTGACCAACTACGAAGTTCCACCGGAAAAACTGGTGATTTATGAGCAAATCACGCGTCCGATCAATGAATATAAGGCCATTGGCCCGCACGTCGCGGTGGCCAAGCGCCTGATGGCGCGTGGTATCAAAGTGAAACCGGGTATGGTTATTGGTTACATCGTGCTGCGTGGCGACGGCCCGATTAGCAAACGTGCGATCAGCATTGAAGAATTTGACCCGCGTAAGCACAAATATGACGCGGAATACTATATCGAGAATCAAGTGCTGCCGGCCGTGGAACGCATTCTGAAAGCTTTCGGCTACAAGCGTGAAGATTTGCGCTGGCAGAAAACCAAACAGGTTGGTCTTGGTGCGTGGATCAAGGTCAAAAAGTCCTAAGGTTGAGGTCTCACCCCCTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCCCCTGAGACGCGTCAATCGAGTTCGTACCTAAGGGCGACACCCCCTAATTAGCCCGGGCGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGATGCCTGGCAGTTCCCTACTCTCGCATGGGGAGTCCCCACACTACCATCGGCGCTACGGCGTTTCACTTCTGAGTTCGGCATGGGGTCAGGTGGGACCACCGCGCTACTGCCGCCAGGCAAACAAGGGGTGTTATGAGCCATATTCAGGTATAAATGGGCTCGCGATAATGTTCAGAATTGGTTAATTGGTTGTAACACTGACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAATATGAGCCATATTCAACGGGAAACGTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGCTTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCACTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGAAAAACAGCGTTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCACTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGCCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAACTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAAGCGGCGCGCCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGAGAGTCAATTCAGGGTGGTGAATATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAGGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGATAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCAGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGACTCATGACCAAAATCCCTTAACGTGAGTTACGCGCGCGTCGTTCCACTGAGCGTCAGACDNA Sequence of wild type P. horikoshii OT3 (SEQ ID NO: 3):Pyrococcus horikoshii DNA Polymerase geneATGATTCTGGACGCTGATTATATTACTGAAGATGGTAAACCGATTATTCGTATTTTTAAAAAAGAAAATGGCGAGTTCAAAGTTGAATATGACCGTAACTTTCGTCCGTACATCTACGCGCTGTTGCGCGACGATAGCGCGATCGATGAGATTAAGAAAATTACCGCGCAGCGTCATGGTAAAGTTGTTCGCATCGTTGAAACCGAGAAAATTCAACGTAAATTCCTGGGCCGCCCAATTGAAGTGTGGAAGCTGTACCTGGAGCATCCGCAAGATGTCCCGGCGATCCGTGACAAGATTCGCGAGCACCCGGCCGTCGTCGACATTTTCGAATACGATATTCCGTTCGCAAAGCGTTACCTGATCGATAAGGGTCTGACCCCGATGGAGGGTAATGAAAAGCTGACGTTCCTGGCTGTCGATATTGAAACGTTGTACCACGAGGGTGAAGAGTTTGGTAAGGGCCCGGTCATTATGATCAGCTACGCGGATGAAGAGGGCGCCAAAGTTATCACGTGGAAAAAAATTGATCTGCCGTACGTTGAAGTTGTGTCCAGCGAGCGCGAGATGATTAAACGCTTGATTCGTGTGATTAAAGAAAAAGATCCAGACGTGATCATTACCTATAATGGTGACAACTTTGACTTTCCGTACTTGCTGAAACGTGCTGAGAAACTGGGTATCAAGCTGTTGCTGGGTCGCGATAATAGCGAGCCGAAGATGCAAAAAATGGGCGATAGCCTGGCAGTCGAGATCAAGGGTCGCATCCACTTTGATCTCTTTCCGGTGATTCGTCGCACGATCAATCTGCCGACCTATACGCTGGAAGCTGTCTACGAGGCAATCTTTGGTAAGCCGAAAGAAAAAGTCTATGCGGACGAAATTGCGAAAGCGTGGGAAACCGGCGAGGGCCTGGAGCGTGTGGCAAAGTACTCTATGGAAGATGCCAAAGTGACCTATGAACTGGGTCGTGAGTTCTTCCCAATGGAAGCCCAGTTGGCGCGCTTGGTGGGCCAACCGGTTTGGGACGTTTCCCGTAGCAGCACCGGTAACCTGGTTGAGTGGTTTCTGTTGCGTAAAGCGTATGAGCGTAATGAACTGGCACCGAACAAGCCTGACGAGAAAGAATATGAACGTCGCCTGCGTGAATCTTACGAGGGTGGTTACGTCAAAGAACCGGAAAAGGGTCTGTGGGAAGGCATCGTGAGCCTGGATTTCCGTAGCCTGTACCCTAGCATCATCATCACGCACAATGTTAGCCCGGACACCCTGAACCGCGAGGGCTGCGAAGAGTACGACGTTGCGCCGAAAGTCGGCCATCGTTTTTGTAAAGACTTCCCTGGTTTCATCCCAAGCCTGCTGGGTCAGCTGCTGGAAGAGAGACAGAAAATTAAAAAACGCATGAAAGAATCGAAAGATCCGGTTGAGAAAAAGCTGCTGGATTACCGCCAGCGTGCCATCAAGATTCTGGCTAACTCATATTATGGCTACTACGGTTATGCTAAAGCGCGTTGGTACTGTAAAGAGTGCGCGGAGTCCGTCACCGCGTGGGGTCGCCAGTATATCGATCTGGTGCGTCGCGAGCTGGAAGCGCGTGGTTTTAAGGTCCTGTACATCGATACTGACGGTCTGTATGCAACCATCCCTGGTGTCAAAGACTGGGAAGAGGTTAAGCGTCGTGCACTGGAATTTGTGGACTATATCAATTCTAAGTTGCCGGGTGTGCTGGAGCTGGAGTACGAAGGCTTCTATGCACGCGGCTTTTTCGTTACGAAAAAGAAATACGCACTGATCGACGAAGAGGGCAAGATTGTGACTCGTGGTCTGGAAATCGTTCGTCGCGACTGGAGCGAGATTGCAAAAGAAACCCAAGCTCGCGTTCTGGAAGCAATCCTGAAACATGGTAACGTCGAAGAAGCCGTCAAGATCGTGAAAGATGTCACCGAAAAGTTGACCAACTACGAAGTTCCACCGGAAAAACTGGTGATTTATGAGCAAATCACGCGTCCGATCAATGAATATAAGGCCATTGGCCCGCACGTCGCGGTGGCCAAGCGCCTGATGGCGCGTGGTATCAAAGTGAAACCGGGTATGGTTATTGGTTACATCGTGCTGCGTGGCGACGGCCCGATTAGCAAACGTGCGATCAGCATTGAAGAATTTGACCCGCGTAAGCACAAATATGACGCGGAATACTATATCGAGAATCAAGTGCTGCCGGCCGTGGAACGCATTCTGAAAGCTTTCGGCTACAAGCGTGAAGATTTGCGCTGGCAGAAAACCAAACAGGTTGGTCTTGGTGCGTGGATCAAGGTCAAAAAGTCCTAAPyrococcus abyssi (SEQ ID NO: 21)MIIDADYITEDGKPIIRIFKKEKGEFKVEYDRTFRPYIYALLKDDSAIDEVKKITAERHGKIVRITEVEKVQKKFLGRPIEVWKLYLEHPQDVPAIREKIREHPAVVDIFEYDIPFAKRYLIDKGLTPMEGNEELTFLAVDIETLYHEGEEFGKGPIIMISYADEEGAKVITWKSIDLPYVEVVSSEREMIKRLVKVIREKDPDVIITYNGDNFDFPYLLKRAEKLGIKLPLGRDNSEPKMQRMGDSLAVEIKGRIHFDLFPVIRRTINLPTYTLEAVYEAIFGKSKEKVYAHEIAEAWETGKGLERVAKYSMEDAKVTFELGKEFFPMEAQLARLVGQPVWDVSRSSTGNLVEWFLLRKAYERNELAPNKPDEREYERRLRESYEGGYVKEPEKGLWEGIVSLDFRSLYPSIIITHNVSPDTLNRENCKEYDVAPQVGHRFCKDFPGFIPSLLGNLLEERQKIKKRMKESKDPVEKKLLDYRQRAIKILANSYYGYYGYAKARWYCKECAESVTAWGRQYIDLVRRELESRGFKVLYIDTDGLYATIPGAKHEEIKEKALKFVEYINSKLPGLLELEYEGFYARGFFVTKKKYALIDEEGKIVTRGLEIVRRDWSEIAKETQAKVLEAILKHGNVDEAVKIVKEVTEKLSKYEIPPEKLVIYEQITRPLSEYKAIGPHVAVAKRLAAKGVKVKPGMVIGYIVLRGDGPISKRAIAIEEFDPKKHKYDAEYYIENQVLPAVERILRAFGYRKEDLKYQKTKQVGLGAWLKF Pyrococcus woesei (SEQ ID NO: 22)MILDVDYITEEGKPVIRLFKKENGKFKIEHDRTFRPYIYALLRDDSKIEEVKKITGERHGKIVRIVDVEKVEKKFLGKPITVWKLYLEHPQDVPTIREKVREHPAVVDIFEYDIPFAKRYLIDKGLIPMEGEEELKILAFDIETLYHEGEEFGKGPIIMISYADENEAKVITWKNIDLPYVEVVSSEREMIKRFLRIIREKDPDIIVTYNGDSFDFPYLAKRAEKLGIKLTIGRDGSEPKMQRIGDMTAVEVKGRIHFDLYHVITRTINLPTYTLEAVYEAIFGKPKEKVYADEIAKAWESGENLERVAKYSMEDAKATYELGKEFLPMEIQLSRLVGQPLWDVSRSSTGNLVEWFLLRKAYERNEVAPNKPSEEEYQRRLRESYTGGFVKEPEKGLWENIVYLDFRALYPSIIITHNVSPDTLNLEGCKNYDIAPQVGHKFCKDIPGFIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYAKARWYCKECAESVTAWGRKYIELVWKELEEKFGFKVLYIDTDGLYATIPGGESEEIKKKALEFVKYINSKLPGLLELEYEGFYKRGFFVTKKRYAVIDEEGKVITRGLEIVRRDWSEIAKETQARVLETILKHGDVEEAVRIVKEVIQKLANYEIPPEKLAIYEQITRPLHEYKAIGPHVAVAKKLAAKGVKIKPGMVIGYIVLRGDGPISNRAILAEEYDPKKHKYDAEYYIENQVLPAVLRILEGFGYRKEDLRYQKTRQVGLTSWLNIKKS[Pyrococcus furiosus] (SEQ ID NO: 23)MILDVDYITEEGKPVIRLFKKENGKFKIEHDRTFRPYIYALLRDDSKIEEVKKITGERHGKIVRIVDVEKVEKKFLGKPITVWKLYLEHPQDVPTIREKVREHPAVVDIFEYDIPFAKRYLIDKGLIPMEGEEELKILAFDIETLYHEGEEFGKGPIIMISYADENEAKVITWKNIDLPYVEVVSSEREMIKRFLRIIREKDPDIIVTYNGDSFDFPYLAKRAEKLGIKLTIGRDGSEPKMQRIGDMTAVEVKGRIHFDLYHVITRTINLPTYTLEAVYEAIFGKPKEKVYADEIAKAWESGENLERVAKYSMEDAKATYELGKEFLPMEIQLSRLVGQPLWDVSRSSTGNLVEWFLLRKAYERNEVAPNKPSEEEYQRRLRESYTGGFVKEPEKGLWENIVYLDFRALYPSIIITHNVSPDTLNLEGCKNYDIAPQVGHKFCKDIPGFIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYAKARWYCKECAESVTAWGRKYIELVWKELEEKFGFKVLYIDTDGLYATIPGGESEEIKKKALEFVKYINSKLPGLLELEYEGFYKRGFFVTKKRYAVIDEEGKVITRGLEIVRRDWSEIAKETQARVLETILKHGDVEEAVRIVKEVIQKLANYEIPPEKLAIYEQITRPLHEYKAIGPHVAVAKKLAAKGVKIKPGMVIGYIVLRGDGPISNRAILAEEYDPKKHKYDAEYYIENQVLPAVLRILEGFGYRKEDLRYQKTRQVGLTSWLNIKKS[Pyrococcus glycovorans] (SEQ ID NO: 24)MILDADYITEDGKPIIRIFKKENGEFKVEYDRNFRPYIYALLKDDSQIDEVKKITAERHGKIVRIVDVEKVKKKFLGRPIEVWKLYFEHPQDVPAIRDKIREHPAVVDIFEYDIPFAKRYLIDKGLIPMEGDEELKLLAFDIETLYHEGEEFAKGPIIMISYADEEGAKVITWKKVDLPYVEVVSSEREMIKRFLKVIREKDPDVIITYNGDSFDLPYLVKRAEKLGIKLPLGRDGSEPKMQRLGDMTAVEIKGRIHFDLYHVIRRTINLPTYTLEAVYEAIFGKPKEKVYAHEIAEAWETGKGLERVAKYSMEDAKVTYELGREFFPMEAQLSRLVGQPLWDVSRSSTGNLVEWYLLRKAYERNELAPNKPDEREYERRLRESYAGGYVKEPEKGLWEGLVSLDFRSLYPSIIITHNVSPDTLNREGCMEYDVAPEVKHKFCKDFPGFIPSLLKRLLDERQEIKRRMKASKDPIEKKMLDYRQRAIKILANSYYGYYGYAKARWYCKECAESVTAWGREYIEFVRKELEEKFGFKVLYIDTDGLYATIPGAKPEEIKRKALEFVEYINAKLPGLLELEYEGFYVRGFFVTKKKYALIDEEGKIITRGLEIVRRDWSEIAKETQAKVLEAILKHGNVEEAVKIVKEVTEKLSKYEIPPEKLVIYEQITRPLHEYKAIGPHVAVAKRLAARGVKVRPGMVIGYIVLRGDGPISKRAILAEEFDPRKHKYDAEYYIENQVLPAVLRILEAFGYRKEDLRWQKTKQTGLTAWLNVKKK Pyrococcus sp. NA2 (SEQ ID NO: 25)MILDADYITEDGKPIIRLFKKENGRFKVEYDRNFRPYIYALLKDDSAIDDVRKITSERHGKVVRVIDVEKVKKKFLGRPIEVWKLYFEHPQDVPAMRDKIREHPAVIDIFEYDIPFAKRYLIDKGLIPMEGNEELTFLAVDIETLYHEGEEFGKGPIIMISYADEEGAKVITWKKIDLPYVEVVANEREMIKRLIKVIREKDPDVIITYNGDNFDFPYLLKRAEKLGMKLPLGRDNSEPKMQRLGDSLAVEIKGRIHFDLFPVIRRTINLPTYTLEAVYEAIFGKQKEKVYPHEIAEAWETGKGLERVAKYSMEDAKVTYELGKEFFPMEAQLARLVGQPLWDVSRSSTGNLVEWYLLRKAYERNELAPNKPDEREYERRLRESYEGGYVKEPERGLWEGIVSLDFRSLYPSIIITHNVSPDTLNKEGCGEYDEAPEVGHRFCKDFPGFIPSLLGSLLEERQKIKKRMKESKDPVERKLLDYRQRAIKILANSFYGYYGYAKARWYCKECAESVTAWGRQYIELVRRELEERGFKVLYIDTDGLYATIPGEKNWEEIKRRALEFVNYINSKLPGILELEYEGFYTRGFFVTKKKYALIDEEGKIVTRGLEIVRRDWSEIAKETQAKVLEAILKHGNVEEAVKIVKEVTEKLSNYEIPVEKLVIYEQITRPLNEYKAIGPHVAVAKRLAAKGIKIKPGMVIGYVVLRGDGPISKRAIAIEEFDGKKHKYDAEYYIENQVLPAVERILKAFGYKREDLRWQKTKQVGLGAWLKVKKS[Pyrococcus sp. ST700] (SEQ ID NO: 26)MILDADYITENGKPIIRLFKKENGKFKVEYDRNFRPYIYALLKDDSAIDDVRKITSERHGKVVRVIDVEKVSKKFLGRPIEVWKLYFEHPQDVPAIRDKIREHPAVIDIFEYDIPFAKRYLIDKGLIPMEGNEELSFLAVDIETLYHEGEEFGKGPIIMISYADEEGAKVITWKKIDLPYVEVVANEREMIKRLVRIIREKDPDIIITYNGDNFDFPYLLKRAEKLGIKLPLGRDNSEPKMQRLGESLAVEIKGRIHFDLFPVIRRTINLPTYTLRTVYEAIFGKPKEKVYPHEIAEAWEIGKGLERVAKYSMEDAKVIYELGKEFFPMEAQLARLVGQPVWDVSRSSTGNLVEWFLLRKAYERNELAPNKPDEKEYEKRLRESYEGGYVKEPEKGLWEGIVSLDFRSLYPSIIITHNVSPDTLNREGCGKYDEAPEVGHRFCKDFPGFIPSLLGDLLEERQKIKKRMKESKDPIEKKLLDYRQRAIKILANSFYGYYGYAKARWYCKECAESVTAWGRQYIELVRRELEERGFKVLYIDTDGLYATIPGEKNWEEIKRKALEFVNYINSKLPGILELEYEGFYTRGFFVTKKKYALIDEEGKIITRGLEIVRRDWSEIAKETQAKVLEAILKHGNVEEAVKIVKEVTEKLSNYEIPVEKLVIYEQITRPLNEYKAIGPHVAVAKRLAAKGIKIKPGMVIGYVLLRGDGPISKRAIAIEEFDGKKHKYDAEYYIENQVLPAVERILKAFGYKKEDLRWQ[Pyrococcus kukulkanii] (SEQ ID NO: 27)MILDADYITEDGKPIIRIFKKENGEFKVEYDRNFRPYIYALLKDDSQIDEVRKITAERHGKIVRIIDAEKVRKKFLGRPIEVWRLYFEHPQDVPAIRDKIREHSAVIDIFEYDIPFAKRYLIDKGLIPMEGDEELKLLAFDIETLYHEGEEFAKGPIIMISYADEEEAKVITWKKIDLPYVEVVSSEREMIKRELKVIREKDPDVIITYNGDSFDLPYLVKRAEKLGIKLPLGRDGSEPKMQRLGDMTAVEIKGRIHFDLYHVIRRTINLPTYTLEAVYEAIFGKPKEKVYAHEIAEAWETGKGLERVAKYSMEDAKVTYELGREFFPMEAQLSRLVGQPLWDVSRSSTGNLVEWYLLRKAYERNELAPNKPDEREYERRLRESYAGGYVKEPEKGLWEGLVSLDFRSLYPSIIITHNVSPDTLNREGCREYDVAPEVGHKFCKDFPGFIPSLLKRLLDERQEIKRKMKASKDPIEKKMLDYRQRAIKILANSYYGYYGYAKARWYCKECAESVTAWGREYIEFVRKELEEKFGFKVLYIDTDGLYATIPGAKPEEIKKKALEFVDYINAKLPGLLELEYEGFYVRGFFVTKKKYALIDEEGKIITRGLEIVRRDWSEIAKETQAKVLEAILKHGNVEEAVKIVKEVTEKLSKYEIPPEKLVIYEQITRPLHEYKAIGPHVAVAKRLAARGVKVRPGMVIGYIVLRGDGPISKRAILAEEFDLRKHKYDAEYYIENQVLPAVLRILEAFGYRKEDLRWQKTKQTGLTAWLNIKKK[Pyrococcus yayanosii] (SEQ ID NO: 28)MILDADYITENGKPVVRIFKKENGEFKVEYDRSFRPYIYALLRDDSAIEDIKKITAERHGKVVRVVEAEKVRKKFLGRPIEVWKLYFEHPQDVPAIREKIREHPAVIDIFEYDIPFAKRYLIDKGLIPMEGNEELKLLAFDIETLYHEGDEFGSGPIIMISYADEKGAKVITWKGVDLPYVEVVSSEREMIKRFLRVIREKDPDVIITYNGDNEDEPYLLKRAEKLGMKLPIGRDGSEPKMQRMGDGFAVEVKGRIHFDIYPVIRRTINLPTYTLEAVYEAVFGRPKEKVYPNEIARAWENCKGLERVAKYSMEDAKVTYELGREFFPMEAQLARLVGQPVWDVSRSSTGNLVEWFLLRKAYERNELAPNRPDEREYERRLRESYEGGYVKEPEKGLWEGIIYLDERSLYPSIIITHNISPDTLNKEGCNSYDVAPKVGHRFCKDFPGFIPSLLGQLLDERQKIKRKMKATIDPIERKLLDYRQRAIKILANSYYGYYGYAKARWYCKECAESVTAWGREYIELVSRELEKRGFKVLYIDTDGLYATIPGSREWDKIKERALEFVKYINARLPGLLELEYEGFYKRGFFVTKKKYALIDEEGKIITRGLEIVRRDWSEIAKETQARVLEAILKEGNLEKAVKIVKEVTEKLSKYEVPPEKLVIYEQITRDLKDYKAVGPHVAVAKRLAARGIKVRPGMVIGYLVLRGDGPISRRAIPAEEFDPSRHKYDAEYYIENQVLPAVLRILEAFGYRKEDLRYQKTRQAGLDAWLKRKASL[Pyrococcus sp. ST04] (SEQ ID NO: 29)MILDADYITEDGKPVIRLFKKENGEFKIEYDRTFKPYIYALLKDDSAIDEVRKVTAERHGKIVRIIDVEKVKKKYLGRPIEVWKLYFEHPQDVPAIREKIREHPAVVEIFEYDIPFAKRYLIDKGIVPMDGDEELKLLAFDIETLYHEGEEFGKGPILMISYADEEGAKVITWKRINLPYVEVVSSEREMIKRELKVIREKDPDVIITYNGDSFDFPYLVKRAEKLGIKLPLGRDGSPPKMQRLGDMNAVEIKGRIHFDLYHVVRRTINLPTYTLEAVYEAIFGKPKEKVYAHEIAEAWETGKGLERVAKYSMEDAQVTYELGKEFFPMEVQLTRLVGQPLWDVSRSSTGNLVEWYLLRKAYERNELAPNKPDEREYERRLRESYAGGYVKEPERGLWENIVYLDFRSLYPSIIITHNVSPDTLNREGCRKYDIAPEVGHKFCKDVEGFIPSLLGHLLEERQKIKRKMKATINPVEKKLLDYRQKAIKILANSYYGYYGYAKARWYCKECAESVTAWGREYIELVRKELEGKFGFKVLYIDTDGLYATIPRGDPAEIKKKALEFVRYINEKLPGLLELEYEGFYRRGFFVTKKKYALIDEEDKIITRGLEIVRRDWSEIAKETQAKVLEAILKEGNVEKAVKIVKEVTEKLMKYEVPPEKLVIYEQITRPLNEYKAIGPHVAVAKRLAAKGVKVRPGMVIGYIVLRGDGPISKRAILAEEYDPRKNKYDAEYYIENQVLPAVLRILEAFGYKKEDLKYQKSRQVGLGAWIKVKK[Pyrococcus sp. GB-D](SEQ ID NO: 30)MILDADYITEDGKPIIRIFKKENGEFKVEYDRNFRPYIYALLKDDSQIDEVRKITAERHGKIVRIIDAEKVRKKFLGRPIEVWRLYFEHPQDVPAIRDKIREHSAVIDIFEYDIPFAKRYLIDKGLIPMEGDEELKLLAFDIETLYHEGEEFAKGPIIMISYADEEEAKVITWKKIDLPYVEVVSSEREMIKRFLKVIREKDPDVIITYNGDSFDLPYLVKRAEKLGIKLPLGRDGSEPKMQRLGDMTAVEIKGRIHFDLYHVIRRTINLPTYTLEAVYEAIFGKPKEKVYAHEIAEAWETGKGLERVAKYSMEDAKVTYELGREFFPMEAQLSRLVGQPLWDVSRSSTGNLVEWYLLRKAYERNELAPNKPDEREYERRLRESYAGGYVKEPEKGLWEGLVSLDFRSLYPSIIITHNVSPDTLNREGCREYDVAPEVGHKFCKDFPGFIPSLLKRLLDERQEIKRKMKASKDPIEKKMLDYRQRAIKILANSYYGYYGYAKARWYCKECAESVTAWGREYIEFVRKELEEKFGFKVLYIDTDGLYATIPGAKPEEIKKKALEFVDYINAKLPGLLELEYEGFYVRGFFVTKKKYALIDEEGKIITRGLEIVRRDWSEIAKETQAKVLEAILKHGNVEEAVKIVKEVTEKLSKYEIPPEKLVIYEQITRPLHEYKAIGPHVAVAKRLAARGVKVRPGMVIGYIVLRGDGPISKRAILAEEFDLRKHKYDAEYYIENQVLPAVLRILEAFGYRKEDLRWQKTKQTGLTAWLNIKKK [SE-1] (SEQ ID NO: 31)MILDADYITEDGKPIIRIFKKENGEFKVEYDRNFRPYIYALLRDDSAIDEIKKITAQRHGKVVRIVETEKIQRKFLGRPIEVWKLYLEHPQDVPAIRDKIREHPAVVDIFEYDIPFAKRYLIDKGLTPMEGNEKLTFLAVAIETLYHEGEEFGKGPVIMISYADEEGAKVITWKKIDLPYVEVVSSEREMIKRLIRVIKEKDPDVIITYNGDNFDFPYLLKRAEKLGIKLLLGRDNSEPKMQKMGDSLAVEIKGRIHFDLFPVIRRTINLPTYTLEAVYEAIFGKPKEKVYADEIAKAWETGEGLERVAKYSMEDAKVTYELGREFFPMEAQLARLVGQPVWDVSRSSTGNLVEWFLLRKAYERNELAPNKPDEKEYERRLRESYEGGYVKEPEKGLWEGIVSLDERSSAVSIIITHNVSPDTLNREGCEEYDVAPKVGHRFCKDFPGFIPSLLGQLLEERQKIKKRMKESKDPVEKKLLDYRQRAIKILANSYYGYYGYAKARWYCKECAESVTAWGRQYIDLVRRELEARGFKVLYIDTDGLYATIPGVKDWEEVKRRALEFVDYINSKLPGVLELEYEGFYARGFFVTKKKYALIDEEGKIVTRGLEIVRRDWSEIAKETQARVLEAILKHGNVEEAVKIVKDVTEKLTNYEVPPEKLVIYEQITRPINEYKAIGPHVAVAKRLMARGIKVKPGMVIGYIVLRGDGPISKRAISIEEFDPRKHKYDAEYYIENQVLPAVERILKAFGYKREDLRWQKTKQVGLGAWIKVKKS

What is claimed is:
 1. A polymerase comprising an amino acid sequence that is at least 80% identical to a continuous 500 amino acid sequence within SEQ ID NO: 1; comprising the following amino acids: an alanine or serine at amino acid position 409 or at an amino acid position functionally equivalent to amino acid position 409; a glycine at amino acid position 410 or at an amino acid position functionally equivalent to amino acid position 410; a proline at amino acid position 411 or at an amino acid position functionally equivalent to amino acid position 411; an alanine at amino acid position 129 or at an amino acid position functionally equivalent to amino acid position 129; an alanine at amino acid position 141 or at an amino acid position functionally equivalent to amino acid position 141; an alanine at amino acid position 143 or at an amino acid position functionally equivalent to amino acid position 143; and an alanine or valine at amino acid position 486 or at an amino acid position functionally equivalent to amino acid position
 486. 2. The polymerase of claim 1, comprising a serine at amino acid position 409 or at an amino acid position functionally equivalent to amino acid position
 409. 3. The polymerase of claim 1, comprising an alanine at amino acid position 409 or at an amino acid position functionally equivalent to amino acid position
 409. 4. The polymerase of claim 1, further comprising a glutamic acid at amino acid position 153 or at an amino acid position functionally equivalent to amino acid position
 153. 5. A polymerase comprising an amino acid sequence that is at least 80% identical to a continuous 500 amino acid sequence within SEQ ID NO: 1; comprising the following amino acids: a serine at amino acid position 409 or an amino acid position functionally equivalent to amino acid position 409; an alanine at amino acid position 410 or at an amino acid position functionally equivalent to amino acid position 410; a proline at amino acid position 411 or at an amino acid position functionally equivalent to amino acid position 411; and an alanine at amino acid position 141 or at an amino acid position functionally equivalent to amino acid position 141; and an alanine at amino acid position 143 or at an amino acid position functionally equivalent to amino acid position
 143. 6. The polymerase of claim 1, further comprising at least one of the following amino acids: an alanine at amino acid position 144 or at an amino acid position functionally equivalent to amino acid position 144; a glutamic acid at amino acid position 153 or at an amino acid position functionally equivalent to amino acid position 153; an alanine at amino acid position 215 or at an amino acid position functionally equivalent to amino acid position 215; an alanine at amino acid position 315 or at an amino acid position functionally equivalent to amino acid position 315; an alanine at amino acid positions 215 and 315 or at amino acid positions functionally equivalent to amino acid positions 215 and 315; a serine at amino acid position 515 or at an amino acid position functionally equivalent to amino acid position 515; a leucine at amino acid position 522 or at an amino acid position functionally equivalent to amino acid position 522; an isoleucine at amino acid position 591 or at an amino acid position functionally equivalent to amino acid position 591; a tryptophan at amino acid position 477 or at an amino acid position functionally equivalent to amino acid position 477; an alanine at amino acid position 477 or at an amino acid position functionally equivalent to amino acid position 477; an alanine at amino acid position 478 or at an amino acid position functionally equivalent to amino acid position 478; a serine at amino acid position 479 or at an amino acid position functionally equivalent to amino acid position 479; a tryptophan at amino acid position 477 or at an amino acid position functionally equivalent to amino acid position 477, and an alanine at amino acid position 478 or an amino acid position functionally equivalent to amino acid position 478; an alanine at amino acid position 477 or at an amino acid position functionally equivalent to amino acid position 477, an alanine at amino acid position 478 or at an amino acid position functionally equivalent to amino acid position 478, and a serine at amino acid position 479 or at an amino acid position functionally equivalent to amino acid position 479; an alanine at amino acid position 603 or at an amino acid position functionally equivalent to amino acid position 603; a leucine at amino acid position 640 or at an amino acid position functionally equivalent to amino acid position 640; a glutamic acid at amino acid position 713 or at an amino acid position functionally equivalent to amino acid position 713; an alanine at amino acid position 714 or at an amino acid position functionally equivalent to amino acid position 714; an alanine at amino acid position 719 or at an amino acid position functionally equivalent to amino acid position 719; an alanine at amino acid position 720 or at an amino acid position functionally equivalent to amino acid position 720; or an alanine at amino acid position 736 or at an amino acid position functionally equivalent to amino acid position
 736. 7. The polymerase of claim 1, further comprising at least one of the following: an alanine at amino acid position 144 or at an amino acid position functionally equivalent to amino acid position 144; a serine at amino acid position 515 or at an amino acid position functionally equivalent to amino acid position 515; an isoleucine at amino acid position 591 or at an amino acid position functionally equivalent to amino acid position 591; an alanine at amino acid position 603 or at an amino acid position functionally equivalent to amino acid position 603; a leucine at amino acid position 640 or at an amino acid position functionally equivalent to amino acid position 640; or a glutamic acid at amino acid position 713 or at an amino acid position functionally equivalent to amino acid position
 713. 8. The polymerase of claim 1, further comprising at least one of the following: an alanine at amino acid position 144 or at an amino acid position functionally equivalent to amino acid position 144; a serine at amino acid position 515 or at an amino acid position functionally equivalent to amino acid position 515; an isoleucine at amino acid position 591 or at an amino acid position functionally equivalent to amino acid position 591; or a leucine at amino acid position 640 or at an amino acid position functionally equivalent to amino acid position
 640. 9. The polymerase of claim 1, further comprising the following: an alanine at amino acid position 144 or at an amino acid position functionally equivalent to amino acid position 144; a valine at amino acid position 486 or at an amino acid position functionally equivalent to amino acid position 486; a serine at amino acid position 515 or at an amino acid position functionally equivalent to amino acid position 515; and an isoleucine at amino acid position 591 or at an amino acid position functionally equivalent to amino acid position
 591. 10. The polymerase of claim 1, wherein the polymerase comprises an amino acid sequence that is at least 90% identical to a continuous 500 amino acid sequence within SEQ ID NO:
 1. 11. The polymerase of claim 1, which exhibits an increased rate of incorporation of modified nucleotides, relative to a control.
 12. The polymerase of claim 1, wherein the polymerase is selected from a Pyrococcus abyssi, Pyrococcus endeavori, Pyrococcus furiosus, Pyrococcus glycovorans, Pyrococcus horikoshii, Pyrococcus kukulkanii, Pyrococcus woesei, Pyrococcus yayanosii, Pyrococcus sp., Pyrococcus sp. 1211, Pyrococcus sp. 121, Pyrococcus sp. 303, Pyrococcus sp. 304, Pyrococcus sp. 312, Pyrococcus sp. 32-4, Pyrococcus sp. 321, Pyrococcus sp. 322, Pyrococcus sp. 323, Pyrococcus sp. 324, Pyrococcus sp. 95-12-1, Pyrococcus sp. AV5, Pyrococcus sp. Ax99-7, Pyrococcus sp. C2, Pyrococcus sp. EX2, Pyrococcus sp. Fla95-Pc, Pyrococcus sp. GB-3A, Pyrococcus sp. GB-D, Pyrococcus sp. GBD, Pyrococcus sp. GI-H, Pyrococcus sp. GI-J, Pyrococcus sp. GIL, Pyrococcus sp. HT3, Pyrococcus sp. JT1, Pyrococcus sp. LMO-A29, Pyrococcus sp. LMO-A30, Pyrococcus sp. LMO-A31, Pyrococcus sp. LMO-A32, Pyrococcus sp. LMO-A33, Pyrococcus sp. LMO-A34, Pyrococcus sp. LMO-A35, Pyrococcus sp. LMO-A36, Pyrococcus sp. LMO-A37, Pyrococcus sp. LMO-A38, Pyrococcus sp. LMO-A39, Pyrococcus sp. LMO-A40, Pyrococcus sp. LMO-A41, Pyrococcus sp. LMO-A42, Pyrococcus sp. M24D13, Pyrococcus sp. MA2.31, Pyrococcus sp. MA2.32, Pyrococcus sp. MA2.34, Pyrococcus sp. MV1019, Pyrococcus sp. MV4, Pyrococcus sp. MV7, Pyrococcus sp. MZ14, Pyrococcus sp. MZ4, Pyrococcus sp. NA2, Pyrococcus sp. NS102-T, Pyrococcus sp. P12.1, Pyrococcus sp. Pikanate 5017, Pyrococcus sp. PK 5017, Pyrococcus sp. ST4, Pyrococcus sp. ST700, Pyrococcus sp. Tc-2-70, Pyrococcus sp. Tc95-7C-I, Pyrococcus sp. TC95-7C-S, Pyrococcus sp. Tc95_6, Pyrococcus sp. V211, Pyrococcus sp. V212, Pyrococcus sp. V221, Pyrococcus sp. V222, Pyrococcus sp. V231, Pyrococcus sp. V232, Pyrococcus sp. V61, Pyrococcus sp. V62, Pyrococcus sp. V63, Pyrococcus sp. V72, Pyrococcus sp. V73, Pyrococcus sp. VB112, Pyrococcus sp. VB113, Pyrococcus sp. VB81, Pyrococcus sp. VB82, Pyrococcus sp. VB83, Pyrococcus sp. VB85, Pyrococcus sp. VB86, Pyrococcus sp. VB93 polymerase, Pyrococcus furiosus DSM 3638, Pyrococcus sp. GE23, Pyrococcus sp. GI-H, Pyrococcus sp. NA2, Pyrococcus sp. ST04, or Pyrococcus sp. ST700 polymerase.
 13. The polymerase of claim 1, wherein the polymerase is a Pyrococcus abyssi or Pyrococcus horikoshii polymerase.
 14. The polymerase according to claim 1, which is capable of incorporating modified nucleotides at reaction temperatures across the range of 40° C. to 80° C.
 15. The polymerase according to claim 10, comprising the following amino acids: an alanine at amino acid position 129, 141, 143, 144, and 409 or at an amino acid position functionally equivalent to amino acid position 129, 141, 143, 144, and 409, a glycine at amino acid position 410 or at an amino acid position functionally equivalent to amino acid position 410, a proline at amino acid position 411 or at an amino acid position functionally equivalent to amino acid position 411, a valine at amino acid position 486 or at an amino acid position functionally equivalent to amino acid position 486, a threonine at amino acid position 515 or at an amino acid position functionally equivalent to amino acid position 515, an isoleucine at amino acid position 591 or at an amino acid position functionally equivalent to amino acid position 591, and a leucine at amino acid position 640 or at an amino acid position functionally equivalent to amino acid position 640; an alanine at amino acid position 129, 141, 143, 144, and 409 or at an amino acid position functionally equivalent to amino acid position 129, 141, 143, 144, and 409, a glycine at amino acid position 410 or at an amino acid position functionally equivalent to amino acid position 410, a proline at amino acid position 411 or at an amino acid position functionally equivalent to amino acid position 411, a leucine at amino acid position 486 or at an amino acid position functionally equivalent to amino acid position 486, a threonine at amino acid position 515 or at an amino acid position functionally equivalent to amino acid position 515, and an isoleucine at amino acid position 591 or at an amino acid position functionally equivalent to amino acid position 591; an alanine at amino acid position 129, 141, 143, 144, and 409 or at an amino acid positon functionally equivalent to amino acid position 129, 141, 143, 144, and 409, a glycine at amino acid position 410 or at an amino acid position functionally equivalent to amino acid position 410, a proline at amino acid position 411 or at an amino acid position functionally equivalent to amino acid position 411, a valine at amino acid position 486 or at an amino acid position functionally equivalent to amino acid position 486, a threonine at amino acid position 515 or at an amino acid position functionally equivalent to amino acid position 515, an isoleucine at amino acid position 591 or at an amino acid position functionally equivalent to amino acid position 591, and an alanine at amino acid position 603 or at an amino acid position functionally equivalent to amino acid position 603; or an alanine at amino acid position 129, 141, 143, 144, and 409 or at an amino acid position functionally equivalent to amino acid position 129, 141, 143, 144, and 409, a glycine at amino acid position 410 or at an amino acid position functionally equivalent to amino acid position 410, a proline at amino acid position 411 or at an amino acid position functionally equivalent to amino acid position 411, a valine at amino acid position 486 or at an amino acid position functionally equivalent to amino acid position 486, a threonine at amino acid position 515 or at an amino acid position functionally equivalent to amino acid position 515, an isoleucine at amino acid position 591 or at an amino acid position functionally equivalent to amino acid position 591, a tryptophan at amino acid position 477 or at an amino acid position functionally equivalent to amino acid position 477; and an alanine at amino acid position 478 or at an amino acid position functionally equivalent to amino acid position
 478. 16. The polymerase of claim 1, further comprising a serine at amino acid position 515 or an amino acid position functionally equivalent to amino acid position
 515. 17. The polymerase of claim 1, comprising an alanine at amino acid position 486 or at an amino acid position functionally equivalent to amino acid position
 486. 18. The polymerase of claim 1, comprising a valine at amino acid position 486 or at an amino acid position functionally equivalent to amino acid position
 486. 19. The polymerase according to claim 1, wherein the polymerase includes the following amino acid substitution mutations relative to SEQ ID NO:1: M129A; D141A; E143A; T144A; L409A; Y410G; A486V; T515S; T591I; K477W; and K478A.
 20. The polymerase according to claim 1, wherein the polymerase includes the following amino acid substitution mutations relative to SEQ ID NO:1: M129A; D141A; E143A; T144A; L409A; Y410G; A486V; T515S; T591I; and G153E.
 21. The polymerase according to claim 1, wherein the polymerase includes the following amino acid substitution mutations relative to SEQ ID NO:1: M129A; D141A; E143A; T144A; L409A; Y410G; A486V; T515S; T591I; K477A; K478A; and L479S.
 22. A polymerase comprising SEQ ID NO: 1; wherein the polymerase comprises the following amino acid substitution mutations relative to SEQ ID NO:1: D141A; E143A; L409A; Y410G; A486V; and T515S.
 23. The polymerase of claim 22, further comprising M129A.
 24. The polymerase of claim 1, wherein the polymerase comprises an amino acid sequence that is at least 99% identical to a continuous 500 amino acid sequence within SEQ ID NO:
 1. 25. The polymerase of claim 1, wherein the polymerase includes the following amino acid substitution mutations relative to SEQ ID NO:1: (a) M129A; D141A; E143A; T144A; L409A; Y410G; A486V; T515S; T591I; A640L; (b) M129A; D141A; E143A; T144A; L409A; Y410G; A486V; T515S; T591I; K477W; K478A; and A640L; or (c) M129A; D141A; E143A; T144A; L409A; Y410G; A486V; T515S; T591I; K603A; and A640L.
 26. A kit comprising the polymerase of claim
 1. 27. A method of incorporating a modified nucleotide into a nucleic acid sequence comprising allowing the following components to interact: (i) a nucleic acid template, (ii) a nucleotide solution, and (iii) a polymerase, wherein the polymerase is a polymerase of claim
 1. 28. The method of claim 27, wherein the polymerase is capable of incorporating a modified nucleotide into a nucleic acid sequence in stringent hybridization conditions.
 29. The method of claim 28, wherein the polymerase is capable of incorporating a modified nucleotide into a nucleic acid sequence at 55° C. to 80° C. 